KR20190142394A - Stable preparations of anti-TIGIT antibodies alone and in combination with programmed kill receptor 1 (PD-1) antibodies and methods of use thereof - Google Patents

Abstract

The present invention provides a stable preparation of an antibody against T cell immunoreceptors (TIGIT) having Ig and ITIM domains, optionally adding an anti-human programmed kill receptor 1 (PD-1) antibody or antigen binding fragment thereof. It relates to a stable formulation containing. Also provided are methods of treating various cancers and chronic infections using the formulations of the present invention.

Description

Stable preparations of anti-TIGIT antibodies alone and in combination with programmed kill receptor 1 (PD-1) antibodies and methods of use thereof

The present invention relates to the preparation of therapeutic antibodies and their use in the treatment of various disorders. In one aspect, the invention relates to an agent comprising an antibody or antigen binding fragment thereof that binds to a T cell immunoreceptor (TIGIT) having an Ig and ITIM domain. In another aspect, the formulation further comprises an anti-human programmed death receptor 1 (PD-1) antibody or antigen binding fragment thereof. Also provided are methods of treating various cancers and chronic infections using the formulations of the present invention.

Cross Reference to Related Application

This application claims the benefit of U.S.S.N 62 / 500,278, filed May 2, 2017, which is hereby incorporated by reference in its entirety.

Reference to the electronically submitted sequence listing

The Sequence Listing in this application is an ASCII formatted sequence listing filed electronically via EFS-Web, file name "24453WOPCT-SEQTXT-01MAY2018.TXT", dated May 1, 2018, size 227 Kb. will be. This sequence listing, submitted via EFS-Web, is part of this specification, the entirety of which is incorporated herein by reference.

Human antibody drugs may differ somewhat in the amino acid sequence of their constant domains, or in their framework sequences within the variable domains, but such antibody drugs typically show the most dramatic difference in CDR sequences. Although antibodies that bind to the same protein, the same polypeptide, or even potentially the same epitope may comprise completely different CDR sequences. Therapeutic antibodies for humans can also be obtained from human germline antibody sequences, such as in humanized antibodies, or from non-human (eg rodent) germline antibody sequences, which further diversify in potential CDR sequences. Induce. Such sequence differences result in different stability in solution and different responsiveness to solution parameters. In addition, small changes in amino acid sequence or variations in one or several amino acid residues can result in dramatically different antibody stability and sensitivity to sequence specific degradation pathways. As a result, the solution conditions necessary to optimize antibody stability are currently unpredictable. Each antibody must be studied individually to determine the optimal solution formulation (Bhambhani et al. (2012) J. Pharm. Sci. 101: 1120).

Antibodies are also relatively high molecular weight proteins (˜150,000 Da), for example when compared to other therapeutic proteins such as hormones and cytokines. As a result, it is frequently necessary to dose the antibody in relatively high weight amounts to achieve the desired molar concentration of drug. In addition, it is usually desirable to administer the antibody drug subcutaneously, because subcutaneous administration is self-administrable. Self-administration allows, for example, to avoid the time and expense associated with visiting a medical facility for intravenous administration. Subcutaneous delivery is actually limited by the volume of solution that can be delivered to the injection site, typically about 1 to 1.5 ml, in a single injection. Subcutaneous self-administration is typically accomplished using a syringe or self-injector prefilled with a liquid solution formulation of the drug rather than a lyophilized form to eliminate the need for the patient to resuspend the drug prior to injection. Antibody drugs must be stable during storage for assured efficacy and consistent dosing, so whatever formulation is selected, they support desirable properties such as high concentration, transparency and acceptable viscosity, and are also acceptable under typical storage conditions. It is important to maintain these properties and drug efficacy over a long storage period.

TIGIT (T cell immunoreceptor with Ig and ITIM domains) is an immunomodulatory receptor mainly expressed on activated T cells and NK cells. TIGIT is also VSIG9; VSTM3; And WUCAM. Its structure shows one extracellular immunoglobulin domain, a type 1 transmembrane region and two ITIM motifs. TIGIT forms part of a costimulatory network consisting of positive (CD226) and negative (TIGIT) immunomodulatory receptors on T cells and ligands expressed on APC (CD155 and CD112).

An important feature in the structure of TIGIT is the presence of an immunoreceptor tyrosine based inhibitory motif (ITIM) in its cytoplasmic tail domain. Like PD-1 and TIGIT, the ITIM domain in the cytoplasmic region of TIGIT recruits tyrosine phosphatases such as SHP-1 and SHP-2, followed by immunoreceptor tyrosine-based activation motifs on T cell receptor (TCR) subunits. (ITAM) is expected to dephosphorylate tyrosine residues. Therefore, ligation of TIGIT by receptors of ligands CD155 and CD112 expressed by tumor cells or TAMS may be responsible for inhibiting TCR signaling and T cell activation, which are essential for increasing effective anti-tumor immunity. Thus, antagonist antibodies specific for TIGIT can inhibit CD155 and CD112 induced T cell response inhibition and enhance anti-tumor immunity.

For example, there is a need for stable formulations of pharmaceutical anti-TIGIT antibodies for the treatment of various cancers and infectious diseases, as well as anti-TIGIT antibodies co-formulated with anti-human PD-1 antibodies. Preferably, the formulation will have a long shelf life and will be stable upon storage and transport, preferably stable for months to years under conditions typical for storage of drugs for self-administration in syringes, ie at refrigerator temperatures. As such, a long term shelf life for the corresponding drug product will be obtained.

In one aspect, the invention provides an anti-TIGIT antibody, or antigen binding fragment thereof, comprising (i) an anti-TIGIT antibody, or antigen binding fragment thereof; (ii) buffers, (iii) non-reducing sugars; (iv) nonionic surfactants; And (v) an agent of an anti-TIGIT antibody, or antigen binding fragment thereof, comprising an antioxidant. In one embodiment, the formulation further comprises an anti-PD-1 antibody, such as pembrolizumab or nivolumab. In another embodiment, the formulation comprises a chelator.

In one embodiment of the invention, the formulation comprises (i) from about 10 mg / ml to about 200 mg / ml of an anti-TIGIT antibody, or antigen binding fragment thereof; (ii) about 5 mM to about 20 mM buffer; (iii) about 6% weight / volume (w / v) to about 8% (w / v) non-reducing sugar; (iv) about 0.01% (w / v) to about 0.10% (w / v) nonionic surfactant; And (v) about 1 mM to about 20 mM antioxidant. In one embodiment, the formulation further comprises an anti-PD-1 antibody, such as pembrolizumab or nivolumab. In another embodiment, the formulation further comprises a chelator. In one embodiment, the pH of the formulation is 4.5-6.5. In certain embodiments, the pH of the formulation is about pH 5.5 to about pH 6.2. In further embodiments, the pH of the formulation is about pH 5.6 to about pH 6.0. In another embodiment, the pH of the formulation is about 5.7. In another embodiment, the pH of the formulation is about 5.8. In another embodiment, the pH of the formulation is about 5.9. In another embodiment, the pH of the formulation is about 6.0. In another embodiment, the pH of the formulation is about 6.1. In another embodiment, the pH of the formulation is about 6.2.

In one embodiment of the formulation, the buffer is L-histidine buffer or sodium acetate, the non-reducing sugar is sucrose, the nonionic surfactant is polysorbate 80, and the antioxidant is methionine, or a pharmaceutically acceptable salt thereof. . In one embodiment, the antioxidant is L-methionine. In another embodiment, the antioxidant is a pharmaceutically acceptable salt of L-methionine, such as, for example, methionine HCl.

In another embodiment, the formulation comprises (i) about 10 mg / ml to about 200 mg / ml of the anti-TIGIT antibody, or antigen binding fragment thereof; (ii) about 5 mM to about 20 mM L-histidine buffer or about 5 mM to about 20 mM sodium acetate buffer; (iii) about 6% w / v to about 8% w / v sucrose; (iv) about 0.01% (w / v) to about 0.10% (w / v) polysorbate 80; And (v) about 1 mM to about 20 mM L-methionine. In another embodiment, the formulation further comprises an anti-PD-1 antibody, such as pembrolizumab or nivolumab. In one embodiment, the formulation further comprises a chelator. In one embodiment, the chelator is present in an amount from about 1 μM to about 50 μM. In one embodiment, the chelator is DTPA. In another embodiment, the chelator is EDTA. In one embodiment, the buffer is L-histidine buffer. In one embodiment, the formulation comprises about 8 mM to about 12 mM L-histidine buffer. In another embodiment, the formulation comprises about 5 mM to about 10 mM of L-methionine. In a further embodiment, the formulation comprises Polysorbate 80 at a weight ratio of approximately 0.02% (w / v). In one embodiment, the anti-TIGIT formulation comprises sucrose in a weight ratio of about 7% (w / v). In any of the above embodiments, the methionine is L-methionine.

In an embodiment of the formulation, the concentration of anti-TIGIT antibody or antigen binding fragment thereof is from about 10 mg / ml to about 100 mg / ml. In another embodiment, the concentration of anti-TIGIT antibody or antigen binding fragment thereof is about 10 mg / ml, 12.5 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, 50 mg / ml, 75 mg / ml or 100 mg / ml. In one embodiment, the concentration of anti-TIGIT antibody or antigen binding fragment thereof is about 20 mg / ml. In one embodiment, the concentration of anti-TIGIT antibody or antigen binding fragment thereof is about 25 mg / ml. In one embodiment, the concentration of anti-TIGIT antibody or antigen binding fragment thereof is about 50 mg / ml. In one embodiment, the concentration of anti-TIGIT antibody or antigen binding fragment thereof is about 75 mg / ml. In one embodiment, the concentration of anti-TIGIT antibody or antigen binding fragment thereof is about 100 mg / ml.

In one aspect, about 20 mg / ml anti-TIGIT antibody or antigen binding fragment thereof, 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% w / v polysorbate 80, and about 10 Provided is a formulation comprising mM L-methionine.

In one aspect, about 25 mg / ml anti-TIGIT antibody or antigen binding fragment thereof, 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% w / v polysorbate 80, and about 10 Provided is a formulation comprising mM L-methionine.

In one aspect, about 50 mg / ml anti-TIGIT antibody or antigen binding fragment thereof, 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% w / v polysorbate 80, and about 10 Provided is a formulation comprising mM L-methionine.

In one aspect, about 75 mg / ml anti-TIGIT antibody or antigen binding fragment thereof, 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% w / v polysorbate 80, and about 10 Provided is a formulation comprising mM L-methionine.

In one aspect, about 100 mg / ml anti-TIGIT antibody or antigen binding fragment thereof, 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% w / v polysorbate 80, and about 10 Provided is a formulation comprising mM L-methionine.

In one aspect of any of the above formulations, the pH of the formulation is from about 5.4 to about 6.2. In another aspect, the pH of the formulation is about 5.5-6.2. In another embodiment, the pH of the formulation is about 5.8-6.1. In another embodiment, the pH is about 5.8. In one embodiment, the pH is 5.9. In another embodiment, the pH is 6.0. In further embodiments, the pH is 6.1.

In one aspect of any of the above formulations, the formulations comprise an anti-PD1 antibody or antigen binding fragment thereof. In one embodiment, the anti-PD1 antibody is pembrolizumab. In another aspect, the anti-PD1 antibody is nivolumab.

In another aspect, the formulation may further comprise a chelator. In one embodiment, the chelator is DTPA. In one embodiment, the chelator is EDTA. In one aspect, the chelator is present in an amount of about 1 μM to about 50 μM. In one embodiment, the formulation comprises about 5 μM of chelator. In one embodiment, the formulation comprises about 10 μM of chelator. In one embodiment, the formulation comprises about 15 μM of chelator. In one embodiment, the formulation comprises about 20 μM of chelator. In one embodiment, the formulation comprises about 25 μM of chelator. In one embodiment, the formulation comprises about 30 μM of chelator. In one embodiment, the formulation comprises about 35 μM of chelator. In one embodiment, the formulation comprises about 40 μM of chelator. In one embodiment, the formulation comprises about 45 μM of chelator. In one embodiment, the formulation comprises about 50 μM of chelator. In one embodiment, the chelating agent is DTPA, which is present in any of the amounts mentioned above. In another embodiment, the chelating agent is EDTA, which is present in any of the amounts mentioned above.

In one embodiment, the formulation is contained in a glass vial. In another embodiment, the formulation is contained in an injection device. In another embodiment, the formulation is a liquid formulation. In one aspect, the formulation is frozen at least below -70 ° C. In another embodiment, the formulation is a reconstituted solution from a lyophilized formulation.

In certain embodiments, the formulation is stable at refrigeration temperature (2-8 ° C.) for at least 3 months, preferably 6 months, and more preferably 1 year, and even more preferably, up to 2 years. In one embodiment of the formulation, after 12 months at 5 ° C., the monomer (%) of the anti-TIGIT antibody is ≧ 90%, as measured by size exclusion chromatography. In another embodiment of the formulation, after 12 months at 5 ° C., the monomer (%) of the anti-TIGIT antibody is ≧ 95% as measured by size exclusion chromatography. In another embodiment of the formulation, after 12 months at 5 ° C., the heavy and light% of anti-TIGIT antibody is ≧ 90%, as measured by reduced CE-SDS. In another embodiment of the formulation, after 12 months at 5 ° C., the heavy and light chain (%) of the anti-TIGIT antibody is ≧ 95% as measured by reduced CE-SDS. In another embodiment of the formulation, after 12 months at 5 ° C., intact IgG (%) of the anti-TIGIT antibody is ≧ 90%, as measured by non-reduced CE-SDS. In another embodiment of the formulation, after 12 months at 5 ° C., intact IgG (%) of the anti-TIGIT antibody is ≧ 95% as measured by non-reduced CE-SDS.

In one aspect of any of the agents described above, the agent is an anti-TIGIT antibody or antigen binding fragment thereof comprising three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs are SEQ ID NOs. CDRL1 or a variant thereof 111, CDRL2 or a variant thereof SEQ ID NO: 112, CDRL3 or a variant thereof, SEQ ID NO: 113, the heavy chain CDRs CDRH1 or a variant thereof, SEQ ID NO: 154 CDRH2 or Anti-TIGIT antibodies or antigen binding fragments thereof, and variants thereof, and CDRH3 of SEQ ID NO: 110 or variants thereof. In one aspect of any of the agents described above, the agent is an anti-TIGIT antibody or antigen binding fragment thereof comprising three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs are CDRL1, sequence of SEQ ID NO: 111 An anti-TIGIT antibody comprising CDRL2 of SEQ ID NO: 112, CDRL3 of SEQ ID NO: 113, and the heavy chain CDRs comprising CDRH1 of SEQ ID NO: 108, CDRH2 of SEQ ID NO: 154, and CDRH3 of SEQ ID NO: 110, or Antigen binding fragments thereof. In another aspect, an agent comprises an anti-TIGIT antibody or antigen-binding fragment thereof comprising a heavy chain variable region or variant thereof comprising SEQ ID NO: 148 and a light chain variable region or variant thereof comprising SEQ ID NO: 152. In another aspect, the formulation comprises an anti-TIGIT antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising SEQ ID NO: 148 and a light chain variable region comprising SEQ ID NO: 152. In one aspect, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a human heavy chain IgG1 constant domain or variant thereof comprising the amino acid sequence of SEQ ID NO: 291, and a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO: 293 or And further variants thereof. In one aspect, the anti-TIGIT antibody or antigen binding fragment thereof further comprises a human heavy chain IgG1 constant domain comprising the amino acid sequence of SEQ ID NO: 291, and a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO: 293 do. In another aspect, the anti-TIGIT antibody or antigen-binding fragment thereof further comprises a human heavy chain IgG4 constant domain comprising the amino acid sequence of SEQ ID NO: 292, and a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO: 293 Include. In another aspect, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a human heavy chain IgG4 constant domain or variant thereof comprising the amino acid sequence of SEQ ID NO: 292, and a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO: 293 Or variants thereof.

In one aspect, the invention provides an antibody comprising (i) an anti-TIGIT antibody, or antigen binding fragment thereof; (ii) an anti-human PD-1 antibody, or antigen binding fragment thereof, (ii) a buffer, (iii) a non-reducing sugar; (iv) nonionic surfactants; And (v) an anti-TIGIT antibody, or antigen-binding fragment thereof, and an anti-human PD-1 antibody, or antigen-binding fragment thereof, comprising an antioxidant. In one embodiment, the combination formulation further comprises a chelator. In one embodiment, the chelator is EDTA. In another embodiment, the chelator is DTPA. In one embodiment of the combination formulation, the ratio of anti-human PD-1 antibody to anti-TIGIT antibody is 1: 2. In one embodiment of the combination formulation, the ratio of anti-human PD-1 antibody to anti-TIGIT antibody is 1: 1. In one embodiment of the combination formulation, the ratio is 2: 1 of anti-human PD-1 antibody to anti-TIGIT antibody.

In one embodiment of the invention, the combination formulation comprises (i) from about 1 mg / ml to about 200 mg / ml anti-TIGIT antibody, or antigen binding fragment thereof; (ii) about 1 mg / ml to about 200 mg / ml anti-human PD-1 antibody (iii) about 5 mM to about 20 mM buffer; (iv) about 6% weight / volume (w / v) to about 8% (w / v) non-reducing sugar; (v) about 0.01% (w / v) to about 0.10% (w / v) nonionic surfactant; And (vi) about 1 mM to about 20 mM antioxidant. In one embodiment, the combination formulation further comprises a chelator. In one embodiment, the chelator is present in an amount from about 1 μM to about 50 μM. In one embodiment, the chelator is DTPA. In another embodiment, the chelator is EDTA. In one embodiment of the combination formulation, the ratio of anti-human PD-1 antibody to anti-TIGIT antibody is 1: 2. In one embodiment of the combination formulation, the ratio of anti-human PD-1 antibody to anti-TIGIT antibody is 1: 1. In one embodiment of the combination formulation, the ratio is 2: 1 of anti-human PD-1 antibody to anti-TIGIT antibody. In one embodiment, the pH of the combination formulation is 4.5-6.5. In certain embodiments, the pH of the formulation is about pH 5.5 to about pH 6.2. In further embodiments, the pH of the formulation is about pH 5.8-6.0.

In one embodiment of the combination formulation, the buffer is L-histidine buffer or sodium acetate buffer, the non-reducing sugar is sucrose, the nonionic surfactant is polysorbate 80 and the antioxidant is L-methionine. In another embodiment, the co-formulations comprise (i) from about 1 mg / ml to about 100 mg / ml of anti-TIGIT antibody, or antigen binding fragment thereof; (ii) about 1 mg / ml to about 100 mg / ml anti-human PD-1 antibody or antigen binding fragment thereof; (iii) about 5 mM to about 20 mM L-histidine or about 5 mM to about 20 mM sodium acetate buffer; (iv) about 6% w / v to about 8% w / v sucrose; (v) about 0.01% w / v to about 0.10% w / v polysorbate 80; And (vi) about 1 mM to about 20 mM L-methionine. In one embodiment, the combination formulation optionally comprises a chelator. In one embodiment, the chelator is present in an amount from about 1 μM to about 50 μM. In one embodiment, the chelator is DTPA. In another embodiment, the chelator is EDTA. In one embodiment of the combination formulation, the buffer is L-histidine buffer. In one embodiment, the combination formulation comprises about 8 mM to about 12 mM L-histidine buffer. In another embodiment, the combination formulation comprises about 5 mM to about 10 mM L-methionine. In further embodiments, the combination formulation comprises Polysorbate 80 at a weight ratio of approximately 0.02% w / v. In one embodiment, the combination formulation comprises sucrose at a weight ratio of about 7% (w / v).

In an embodiment of the combination formulation, the concentration of the anti-TIGIT antibody or antigen binding fragment thereof is about 1 mg / ml to about 100 mg / ml. In an embodiment of the combination formulation, the concentration of anti-TIGIT antibody or antigen binding fragment thereof is about 10 mg / ml to about 100 mg / ml. In another embodiment, the concentration of anti-TIGIT antibody or antigen binding fragment thereof is about 10 mg / ml. In another embodiment, the concentration of anti-TIGIT antibody or antigen binding fragment thereof is about 12.5 mg / ml. In another embodiment, the concentration of anti-TIGIT antibody or antigen binding fragment thereof is about 20 mg / ml. In another embodiment, the concentration of anti-TIGIT antibody or antigen binding fragment thereof is about 25 mg / ml. In another embodiment, the concentration of anti-TIGIT antibody or antigen binding fragment thereof is about 50 mg / ml. In another embodiment, the concentration of anti-TIGIT antibody or antigen binding fragment thereof is about 75 mg / ml. In another embodiment, the concentration of anti-TIGIT antibody or antigen binding fragment thereof is about 100 mg / ml, or 100 mg / ml.

In some embodiments of the combination formulation, the concentration of anti-human PD-1 antibody is about 1 mg / ml to about 100 mg / ml. In one embodiment of the combination formulation, the concentration of anti-human PD-1 antibody is about 10 mg / ml to about 100 mg / ml. In another embodiment, the concentration of anti-human PD-1 antibody is 20 mg / ml. In another embodiment, the concentration of anti-human PD-1 antibody is 25 mg / ml.

In one embodiment, the combination formulation is about 20 mg / ml anti-PD1 antibody, about 20 mg / ml anti-TIGIT antibody, 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% w / v polysorbate 80, and about 10 mM L-methionine.

In one embodiment, the combination formulation is about 25 mg / ml anti-PD1 antibody, about 25 mg / ml anti-TIGIT antibody, 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% w / v polysorbate 80, and about 10 mM L-methionine.

In one embodiment, the combination formulation is about 50 mg / ml anti-PD1 antibody, about 50 mg / ml anti-TIGIT antibody, 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% w / v polysorbate 80, and about 10 mM L-methionine.

In one aspect of any of the formulations described above, the formulation is an anti-TIGIT antibody or antigen binding fragment thereof comprising three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs are CDRL1 of SEQ ID NO: 111. Or a variant thereof, CDRL2 of SEQ ID NO: 112 or a variant thereof, CDRL3 of SEQ ID NO: 113 or a variant thereof, wherein the heavy chain CDRs comprise CDRH1 of SEQ ID NO: 108 or a variant thereof, CDRH2 of SEQ ID NO: 154, or a variant thereof, And an anti-TIGIT antibody or antigen binding fragment thereof comprising CDRH3 of SEQ ID NO: 110 or a variant thereof. In one aspect of any of the agents described above, the agent is an anti-TIGIT antibody or antigen binding fragment thereof comprising three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs are CDRL1, sequence of SEQ ID NO: 111. An anti-TIGIT antibody comprising CDRL2 of SEQ ID NO: 112, CDRL3 of SEQ ID NO: 113, and the heavy chain CDRs comprising CDRH1 of SEQ ID NO: 108, CDRH2 of SEQ ID NO: 154, and CDRH3 of SEQ ID NO: 110, or Antigen binding fragments thereof. In another aspect, an agent comprises an anti-TIGIT antibody or antigen-binding fragment thereof comprising a heavy chain variable region or variant thereof comprising SEQ ID NO: 148 and a light chain variable region or variant thereof comprising SEQ ID NO: 152. In another aspect, the formulation comprises an anti-TIGIT antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising SEQ ID NO: 148 and a light chain variable region comprising SEQ ID NO: 152. In one aspect, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a human heavy chain IgG1 constant domain or variant thereof comprising the amino acid sequence of SEQ ID NO: 291, and a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO: 293 or And further variants thereof. In one aspect, the anti-TIGIT antibody or antigen binding fragment thereof further comprises a human heavy chain IgG1 constant domain comprising the amino acid sequence of SEQ ID NO: 291, and a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO: 293 do. In another aspect, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a human heavy chain IgG4 constant domain or variant thereof comprising the amino acid sequence of SEQ ID NO: 292, and a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO: 293 Or variants thereof. In another aspect, the anti-TIGIT antibody or antigen-binding fragment thereof further comprises a human heavy chain IgG4 constant domain comprising the amino acid sequence of SEQ ID NO: 292, and a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO: 293 Include.

In one aspect of any of the agents described above, the anti-human PD-1 antibody or antigen binding fragment thereof comprises three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs comprise CDRL1 of SEQ ID NO: 1 or A variant thereof, CDRL2 of SEQ ID NO: 2 or a variant thereof, CDRL3 of SEQ ID NO: 3, or a variant thereof, wherein the heavy chain CDRs comprise CDRH1 of SEQ ID NO: 6 or a variant thereof, CDRH2 of SEQ ID NO: 7, or a variant thereof, and CDRH3 of SEQ ID NO: 8 or variant thereof. In one aspect of any of the agents described above, the anti-human PD-1 antibody or antigen binding fragment thereof comprises three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs comprise CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, CDRL3 of SEQ ID NO: 3, and the heavy chain CDRs include CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8. In another aspect, an agent comprises an anti-human PD1 antibody or antigen binding fragment thereof comprising a light chain variable region or variant thereof comprising SEQ ID NO: 4 and a heavy chain variable region comprising SEQ ID NO: 9 or a variant thereof. . In another aspect, the formulation comprises an anti-human PD1 antibody or antigen binding fragment thereof comprising a light chain variable region comprising SEQ ID NO: 4 and a heavy chain variable region comprising SEQ ID NO: 9. In another aspect, the formulation comprises an anti-human PD1 antibody or antigen binding fragment thereof comprising a light chain comprising SEQ ID NO: 5, and a heavy chain comprising SEQ ID NO: 10. In another aspect, the formulation comprises an anti-human PD1 antibody or antigen binding fragment thereof comprising a light chain comprising SEQ ID NO: 5 or a variant thereof, and a heavy chain comprising SEQ ID NO: 10 or a variant thereof. In one aspect of any of the agents described above, the anti-human PD-1 antibody or antigen binding fragment thereof is pembrolizumab. In another aspect, the anti-human PD-1 antibody or antigen binding fragment thereof is nivolumab.

In one aspect of any of the combination formulations described above, the formulation is (i) an anti-TIGIT antibody or antigen binding fragment thereof comprising three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs are SEQ ID NO: 111 CDRL1 of SEQ ID NO: 112, CDRL2 of SEQ ID NO: 112, CDRL3 of SEQ ID NO: 113, and the heavy chain CDRs include CDRH1 of SEQ ID NO: 108, CDRH2 of SEQ ID NO: 154, and CDRH3 of SEQ ID NO: 110. -TIGIT antibody or antigen-binding fragment thereof, and (ii) an anti-human PD-1 antibody or antigen-binding fragment thereof comprising three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs are CDRL1 of SEQ ID NO: 1. , CDRL2 of SEQ ID NO: 2, CDRL3 of SEQ ID NO: 3, wherein the heavy chain CDRs comprise CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8 PD-1 antibody or antigen-binding fragment thereof It should.

In one aspect of any of the above complex formulations, the formulation comprises (i) an anti-TIGIT antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising SEQ ID NO: 148 and a light chain variable region comprising SEQ ID NO: 152, And (ii) an anti-human PD1 antibody or antigen binding fragment thereof comprising a light chain variable region comprising SEQ ID NO: 4 and a heavy chain variable region comprising SEQ ID NO: 9.

In another aspect of any of the combination formulations, the formulation further comprises (i) a heavy chain variable region comprising SEQ ID NO: 148 and further comprising a human heavy chain IgG1 constant domain comprising the amino acid sequence of SEQ ID NO: 291 An anti-TIGIT antibody or antigen binding fragment thereof comprising a light chain variable region comprising SEQ ID NO: 152 and further comprising a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO: 293, and (ii) An anti-human PD1 antibody or antigen binding fragment thereof comprising a light chain comprising SEQ ID NO: 5, and a heavy chain comprising SEQ ID NO: 10.

In another aspect of any of the combination formulations, the formulation further comprises (i) a heavy chain variable region comprising SEQ ID NO: 148 and further comprising a human heavy chain IgG1 constant domain comprising the amino acid sequence of SEQ ID NO: 292 An anti-TIGIT antibody or antigen binding fragment thereof comprising a light chain variable region comprising SEQ ID NO: 152 and further comprising a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO: 293, and (ii) An anti-human PD1 antibody or antigen binding fragment thereof comprising a light chain comprising SEQ ID NO: 5, and a heavy chain comprising SEQ ID NO: 10.

In one embodiment of any of the formulations described above, the formulation is contained in a glass vial. In another embodiment, the formulation is contained in an injection device. In another embodiment, the formulation is a liquid formulation. In one aspect, the formulation is frozen at least below -70 ° C. In another embodiment, the formulation is a reconstituted solution from a lyophilized formulation.

The present invention provides a method of treating chronic infection or cancer in a mammalian subject (eg, a human) in need thereof, comprising administering an effective amount of an anti-TIGIT agent or a combination formulation described herein. To provide.

1 shows the pH stability of the formulation over 9 months at various storage conditions.
2 shows the polysorbate 80 concentration stability of the formulation over 9 months at various storage conditions.
3 shows the efficacy by ELISA stability data for formulations over 9 months at various storage conditions.
FIG. 4 shows monomers (%) by UP-SEC stability data for formulations over 9 months at various storage conditions.
FIG. 5 shows high molecular weight (HMW) species (%) by UP-SEC stability data for formulations over 9 months at various storage conditions.
FIG. 6 shows low molecular weight (LMW) species (%) by UP-SEC stability data for formulations over 9 months at various storage conditions.
FIG. 7 shows the purity (%) of heavy + light chain by CE-SDS reducible stability data for formulations over 9 months at various storage conditions.
FIG. 8 shows the purity (in%) of intact IgG by CE-SDS non-reducing stability data for formulations over 9 months at various storage conditions.

In one aspect, the invention provides an agent comprising an anti-TIGIT antibody comprising methionine and an antigen binding fragment thereof. Also provided are complex preparations of anti-TIGIT antibodies or antigen binding fragments thereof, and anti-human PD-1 antibodies or antigen binding fragments thereof comprising methionine. In each case, the formulation and the combination formulation optionally include a chelating agent.

I. Definitions and Abbreviations

As used throughout this specification and the appended claims, the following abbreviations apply:

API Active Pharmaceutical Ingredients

CDR Complementarity determining regions in immunoglobulin variable regions, defined using the Kabat numbering system, unless otherwise specified

CHO Chinese Hamster Ovary

CI confidence interval

DTPA diethylenetriaminepentaacetic acid

Concentrations that result in EC50 50% potency or binding

ELISA enzyme-linked immunosorbent assay

FFPE formalin fixed, paraffin embedded

FR framework area

HRP Horseradish Peroxidase

HNSCC Head and Neck Squamous Cell Carcinoma

Concentrations resulting in 50% inhibition of IC50

IgG immunoglobulin G

IHC immunohistochemistry or immunohistochemistry

mAb monoclonal antibodies

MES 2- (N-morpholino) ethanesulfonic acid

NCBI National Center for Biotechnology Information

NSCLC non-small cell lung cancer

PCR polymerase chain reaction

PD-1 Programmed Death 1 (also known as Programmed Cell Death-1 and Programmed Death Receptor 1)

PD-L1 Programmed Cell Death 1 Ligand 1

PD-L2 Programmed Cell Death 1 Ligand 2

PS80 Polysorbate 80

TNBC Triple Negative Breast Cancer

V _H immunoglobulin heavy chain variable region

VK immunoglobulin kappa light chain variable region

V _L immunoglobulin light chain variable region

v / v volume / volume

WFI Injection Water

w / v weight / volume.

As such, specific technical and scientific terms are specifically defined below, in order that the present invention may be more readily understood. Unless specifically defined elsewhere herein, all other technical and scientific terms used herein have the meanings that are commonly understood by one of ordinary skill in the art to which this invention belongs.

Throughout this specification and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

Reference to “or” indicates one or both of the possibilities unless the context clearly indicates one of the specified possibilities. In some cases, “and / or” is used to highlight one or both of the possibilities.

As used herein, “treating” or “treating” cancer refers to a subject suffering from an immune or cancerous condition or diagnosed with a cancer or pathogenic infection (eg, viral, bacterial, fungal infection). Administering the agents of the invention to achieve at least one positive therapeutic effect, such as, for example, reducing cancer cell numbers, reducing tumor size, decreasing the rate of cancer cell infiltration into surrounding organs, or decreasing the rate of tumor metastasis or tumor growth Means that. "Treatment" refers to: inducing / increasing an anti-tumor immune response, stimulating an immune response against pathogens, toxins and / or self antigens, stimulating an immune response to viral infections, one or more tumor markers Reducing the number, inhibiting the growth or survival of tumor cells, removing one or more cancerous lesions or tumors, or reducing their size, reducing the level of one or more tumor markers, the severity of the cancer or Improving or reducing the duration, or extending the survival of the patient as compared to the expected survival in similar untreated patients.

"Immune condition" or "immune disorder" includes, for example, pathological inflammation, inflammatory disorders, and autoimmune disorders or diseases. "Immune condition" also refers to cancer, tumors, and angiogenesis, including infections, persistent infections, and proliferative conditions, such as infections, tumors, and cancers that resist eradication by the immune system. "Cancerous conditions" include, for example, cancer, cancer cells, tumors, angiogenesis, and precancerous conditions such as dysplasia.

Positive therapeutic effects in cancer can be measured in a number of ways (see WA Weber, J. Nucl . Med . 50: 1S-10S (2009)). For example, with respect to tumor growth inhibition, according to the NCI standard, T / C ≦ 42% is the minimum level of anti-tumor activity. T / C <10% is considered to be a high anti-tumor activity level, with T / C (%) = median tumor volume of treated / median tumor volume of control × 100. In some embodiments, the treatment achieved by administration of an agent of the invention is any of progression free survival (PFS), disease free survival (DFS) or overall survival (OS). PFS, also referred to as “time to tumor progression”, indicates the length of time during which cancer does not grow during and after treatment, as well as the amount of time a patient experiences complete remission or partial remission, as well as a stable lesion Include the amount of time you have experienced. DFS refers to the length of time that a patient remains disease free during and after treatment. OS refers to prolongation in life expectancy as compared to naive or untreated individuals or patients. While embodiments of the formulations, methods of treatment, and uses of the invention may not be effective in achieving a positive therapeutic effect in all patients, any statistical assay known in the art, such as Student's t test, chi ² -black, U-test according to Mann and Whitney, Kruskal-Wallis test (H-black), Jonckheere-Terpstra-black and As determined by the Wilcoxon-test, it should be effective in a statistically significant number of subjects.

The term "patient" (alternatively referred to herein as "subject" or "individual") refers to mammals (eg, rats, mice, dogs, cats, rabbits) and most preferred that can be treated with an agent of the present invention. To human. In some embodiments, the patient is an adult patient. In other embodiments, the patient is a pediatric patient.

"Antibody" refers to any form of antibody that exhibits the desired biological activity. Thus, it is used in its broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, humanized, fully human antibodies, chimeric antibodies. A “parent antibody” is an antibody obtained by exposing the immune system to an antigen prior to modification of the antibody for its intended use, such as humanization of the antibody for use as an antibody for human treatment.

In general, basic antibody structural units include tetramers. Each tetramer comprises two identical pairs of polypeptide chains, each pair having one "light chain" (about 25 kDa) and one "heavy chain" (about 50-70 kDa). The amino-terminal portion of each chain comprises a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The variable region of each light / heavy chain pair forms the antibody binding site. Thus, intact antibodies generally have two binding sites. The carboxy-terminal portion of the heavy chain may define a constant region that is primarily responsible for effector function. Typically, human light chains are classified as kappa and lambda light chains. In addition, human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon and define the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of more than about 10 amino acids. In general, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)).

Typically, the variable domains of both heavy and light chains comprise three hypervariable regions, also called complementarity determining regions (CDRs), located within relatively conserved framework regions (FRs). CDRs are typically aligned by framework regions, which make it possible to bind to specific epitopes. In general, both the light and heavy chain variable domains include FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, from the N-terminus to the C-terminus. Assigning amino acids to each domain is generally described in Sequences of Proteins of Immunological Interest , Kabat, et al .; National Institutes of Health, Bethesda, Md. ; 5 ^th ed .; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot . Chem . 32: 1-75; Kabat, et al ., (1977) J. Biol . Chem . 252: 6609-6616; Chothia, et al ., (1987) J Mol . Biol . 196: 901-917 or Chothia, et al ., (1989) Nature 342: 878-883.

An antibody that "specifically binds" to a given target protein is an antibody that exhibits preferential binding to that target over other proteins, but this specificity does not require absolute binding specificity. An antibody is considered to be "specific" to its intended target when its binding determines the presence of a target protein in a sample, without producing unwanted results such as, for example, false positives. Antibodies or binding fragments thereof useful in the present invention are at least 2 times larger, preferably at least 10 times larger, more preferably at least 20 times larger, most preferably at least 100 than affinity with non-target proteins. It will bind to the target protein with twice as much affinity. As used herein, if an antibody binds to a polypeptide comprising a given amino acid sequence, such as the amino acid sequence of a mature human TIGIT or human PD-1, but does not bind to a protein that lacks the sequence, the antibody binds to that sequence. Referred to specifically binding to the polypeptide comprising.

A “chimeric antibody” is one in which a portion of the heavy and / or light chain is derived from a particular species (eg, a human), is identical or homologous to the corresponding sequence of an antibody belonging to a particular antibody class or subclass, and the remaining chain (s). Refers to antibodies that are identical or homologous to the corresponding sequence of an antibody derived from another species (eg, a mouse) or belonging to another antibody class or subclass, as well as fragments of such antibodies as long as they exhibit the desired biological activity do.

As used herein, “co-formulated” or “combined formulation” or “combination” or “co-formulated” is not formulated separately, stored, then mixed prior to administration, or administered separately, Refers to at least two different antibodies or antigen-binding fragments thereof formulated together and stored in a single vial or vessel (eg, an injection device) as a combined product. In one embodiment, the combination preparation contains two different antibodies or antigen binding fragments thereof.

The term "pharmaceutically effective amount" or "effective amount" means an amount of therapeutic composition or agent introduced into a patient sufficient to treat a disease or condition. Those skilled in the art will understand that they may vary depending on the characteristics of the patient, such as age, weight, and the like.

When modifying the amount of a substance or composition (eg, mM, or M), the ratio of formulation components (v / v or w / v), the pH of a solution / formulation, or the value of a parameter characterizing a step in a method, and the like. The term "about" refers to, for example, through typical methods of measurement, handling and sampling involving the manufacture, characterization and / or use of a substance or composition; Through mechanical errors in the method; It refers to variations in the quantity that can occur through differences in the manufacture, source, or purity of ingredients used to prepare or use the composition, or to perform the method. In certain embodiments, “about” can mean a variation of ± 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%.

As used herein, “x% (w / v)” is equal to x g / 100 ml (eg 5% w / v is 50 mg / ml).

The formulations of the present invention comprise antibodies and fragments thereof that are biologically active when reconstituted or in liquid form.

The terms "cancer", "cancerous," or "malignant" refer to or describe the physiological conditions in mammals that are typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, leukemia, blastoma and sarcoma. More specific examples of the cancer include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, gastrointestinal cancer (gastrointestinal cancer), renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, Lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondroma, neuroblastoma, pancreatic cancer, glioblastoma, cervical cancer, brain cancer, gastric cancer, bladder cancer, hepatocellular cancer, breast cancer, colon carcinoma , And head and neck cancer.

"Chothia" is described in Al-Lazikani et al. , JMB 273: 927-948 (1997).

As used herein, “Kabat” refers to an immunoglobulin alignment and numbering scheme pioneered by Elvin A. Kabat ((1991) Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.).

As used herein, “growth inhibitor” refers to a compound or composition that inhibits the growth of cells in vitro or in vivo, particularly cancer cells that overexpress any of the genes identified herein. Thus, growth inhibitory means to significantly reduce the proportion of cells overexpressing the gene in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at stages other than S phase), such as agents that induce G1 arrest and M arrest. Classical M blockers include vinca (vincristine and vinblastine) taxanes, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, and etoposide. The agent that stops G1 also diffuses to S phase arrest, such as, for example, DNA alkylating agents such as dacarbazine, mechlorethamine, and cisplatin. For further information, see The Molecular Basis of Cancer , Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogens, and antineoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995).

The terms “TIGIT binding fragment,” “its antigen binding fragment,”, “its binding fragment” or “its fragment” bind to an antigen (human TIGIT) and inhibit its activity (eg, human TIGIT is a natural ligand thereof). Fragments or derivatives of the antibody that still substantially retain their biological activity). Thus, the term “antibody fragment” or TIGIT binding fragment refers to a portion of a full length antibody, generally its antigen binding region or variable region. Examples of TIGIT antibody fragments include Fab, Fab ', F (ab') ₂ , and Fv fragments. Typically, the binding fragment or derivative retains at least 10% of its TIGIT inhibitory activity. In some embodiments, although any binding fragment having sufficient affinity to exert the desired biological effect will be useful, the binding fragment or derivative may be at least 25%, 50%, 60%, 70%, 80 of its TIGIT inhibitory activity. Maintain%, 90%, 95%, 99% or 100% (or more). In some embodiments, the antigen binding fragment is at least 2 times larger, preferably at least 10 times larger, more preferably at least 20 times larger, and most preferably, at least 100 times greater than affinity with an unrelated antigen. Binds to its antigen with greater affinity. In one embodiment, the antibody has an affinity greater than about 10 ⁹ L / mol, eg, as determined by Scatchard analysis (Munsen et al. (1980) Analyt . Biochem . 107: 220-239). It is also intended that a TIGIT binding fragment may comprise variants having conservative amino acid substitutions that do not substantially alter their biological activity.

The terms “PD-1 binding fragment,” “its antigen binding fragment,”, “its binding fragment” or “its fragment” bind to an antigen (human PD-1) and inhibit its activity (eg, PD- 1 comprises fragments or derivatives of antibodies that still substantially retain their biological activity (which blocks binding to PDL1 and PDL2). Thus, the term “antibody fragment” or PD-1 binding fragment refers to a portion of a full length antibody, generally its antigen binding region or variable region. Examples of antibody fragments include Fab, Fab ', F (ab') ₂ , and Fv fragments. Typically, the binding fragment or derivative retains at least 10% of its PD-1 inhibitory activity. In some embodiments, although any binding fragment having sufficient affinity to exert the desired biological effect will be useful, the binding fragment or derivative may be at least 25%, 50%, 60%, 70% of its PD-1 inhibitory activity. , 80%, 90%, 95%, 99% or 100% (or more). In some embodiments, the antigen binding fragment is at least 2 times larger, preferably at least 10 times larger, more preferably at least 20 times larger, and most preferably, at least 100 times greater than affinity with an unrelated antigen. Binds to its antigen with greater affinity. In one embodiment, the antibody has an affinity greater than about 10 ⁹ L / mol, eg, as determined by Scatchard analysis (Munsen et al. (1980) Analyt . Biochem . 107: 220-239). In addition, PD-1 binding fragments are intended to include variants having conservative amino acid substitutions that do not substantially alter their biological activity.

"Human antibody" refers to an antibody comprising only human immunoglobulin protein sequences. When human antibodies are produced in mice, mouse cells, or hybridomas derived from mouse cells, they may contain murine carbohydrate chains. Similarly, "mouse antibody" or "rat antibody" refers to an antibody comprising only mouse or rat immunoglobulin sequences, respectively.

"Humanized antibody" refers to a form of an antibody containing sequences from non-human (eg, murine) antibodies as well as human antibodies. The antibody contains a minimal sequence derived from non-human immunoglobulin. In general, humanized antibodies will comprise substantially at least one, and typically both variable domains, where all or substantially all hypervariable loops correspond to those of non-human immunoglobulins and all or substantially all The FR region is of human immunoglobulin sequence. Humanized antibodies will also optionally include at least a portion of an immunoglobulin constant region (Fc), typically of human immunoglobulin. Although specific amino acid substitutions may also be included to increase affinity, increase stability of humanized antibodies, or for other reasons, the humanized form of rodent antibodies will generally comprise the same CDR sequences of the parental rodent antibody.

Antibodies of the invention also include antibodies in which the Fc region has been modified (or blocked) to provide altered effector function. See, for example, US Pat. No. 5,624,821; WO2003 / 086310; WO2005 / 120571; WO2006 / 0057702; Presta (2006) Adv . Drug Delivery Rev. 58: 640-656. Such modifications can be used to enhance or inhibit various responses of the immune system and possibly have beneficial effects in diagnosis and therapy. Alterations of the Fc region include amino acid mutations (substitution, deletion and insertion), glycosylation or deglycosylation, and multiple Fc additions. Variations on the Fc may also alter the antibody half-life in the therapeutic antibody, with longer half-lives accompanied by increased convenience and reduced dosing frequency, while reducing substance use. Presta (2005) J. Allergy Clin . Immunol . 116: 731 at 734-35.

"Fully human antibody" refers to an antibody comprising only human immunoglobulin proteins. When fully human antibodies are produced in mice, mouse cells, or hybridomas derived from mouse cells, they may contain murine carbohydrate chains. Similarly, a “mouse antibody” refers to an antibody each containing only mouse immunoglobulin sequences. Fully human antibodies can be produced in humans, in transgenic animals having human immunoglobulin germline sequences, by phage display or by other molecular biological methods.

"Hypervariable region" refers to an amino acid residue of an antibody that is responsible for antigen-binding. Hypervariable regions include amino acid residues from the “complementarity determining regions” or “CDRs” (eg, 24-34 (CDRL1), 50-56 (CDRL2) and 89- in the light chain variable domain, as determined by the Kabat numbering system). 97 (CDRL3) and residues 31-35 (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3) ((Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Health Service, National Institutes of Health, Bethesda, Md.), And / or residues from "hypervariable loops" (ie, 26-32 (L1), 50-52 (L2) and 91-96 in the light chain variable domains). (L3) and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain (Chothia and Lesk (1987) J. Mol. Biol . 196: 901-917). As used herein, the term “framework” or “FR” residues refers to those variable domain residues other than the hypervariable region residues defined herein as CDR residues CDRs and FR residues are described in Kabat. et al. (1987) Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda Md.

"Conservatively modified variants" or "conservative substitutions" refer to amino acid substitutions known to those of skill in the art and, even in essential regions of the polypeptide, generally do not alter the biological activity of the resulting molecule. It can be done without. Preferably said exemplary substitutions are made as described in Table 1 below:

TABLE 1

Exemplary Conservative Amino Acid Substitutions

In addition, one of ordinary skill in the art generally recognizes that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter the biological activity. See, eg, Watson et al. (1987) Molecular Biology of the Gene , The Benjamin / Cummings Pub. Co., p. 224 (4th Edition).

As used throughout the specification and claims, the phrase “consist essentially of” or variations, such as “consist essentially of” or “consist essentially of” any of the mentioned elements or The inclusion of a group of elements, and the optional inclusion of other elements that are similar or different in nature to the mentioned elements, and that do not substantially change the basic or novel properties of the specified dosage regimen, method or composition. By way of non-limiting example, a binding compound consisting essentially of the mentioned amino acid sequence may also include one or more amino acids (including substitution of one or more amino acid residues) that do not essentially affect the properties of the binding compound.

“Comprising,” or variations, such as “comprise,” “comprises,” or “consists of,” unless stated otherwise in the context to express a language or necessary implication, Used throughout the claims in a comprehensive sense, ie, specifying the presence of the stated features, but not excluding the presence or addition of additional features that may substantially enhance the operation or usefulness of any embodiment of the present invention. do.

"Isolated antibody" and "isolated antibody fragment" refer to the state of purification, and in this context, the named molecule refers to other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other substances such as cell debris and Means substantially no growth medium. In general, the term “isolated” refers to the completeness of such a substance, unless such substance, or water, buffer, or salt is present in an amount that substantially interferes with the experimental or therapeutic use of the binding compound as described herein. It is not intended to refer to the absence or absence of water, buffers, or salts.

As used herein, “monoclonal antibody” or “mAb” or “Mab” refers to a population of substantially homologous antibodies, ie, antibody molecules comprising the population may be present in naturally occurring naturally occurring traces. Same in amino acid sequence except mutation. In contrast, conventional (polyclonal) antibody preparations typically include a number of different antibodies with different amino acid sequences in their variable domains, particularly their CDRs, which are often specific for different epitopes. The modifier “monoclonal” characterizes the antibody as obtained from a substantially homogenous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention are described in Kohler et al. (1975) Nature 256: 495, which may be prepared by the hybridoma method described first, or by recombinant DNA methods (see, eg, US Pat. No. 4,816,567). "Monoclonal antibodies" are also described, eg, in Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol . Biol . 222: 581-597 can be isolated from phage antibody libraries using the techniques described. See also, Presta (2005) J. Allergy Clin . Immunol . 116: 731.

"Tumor" when applied to a subject diagnosed with or suspected of having cancer refers to a malignant or potential malignant neoplasm or tissue mass of any size and includes primary tumors and secondary neoplasms. Solid tumors are usually abnormal growths or masses of tissue that do not contain sac or liquid areas. Several types of solid tumors are named for the cell types that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemia (blood cancer) generally does not form a solid tumor (National Cancer Institute, Dictionary of Cancer Terms).

The term "tumor size" refers to the total size of a tumor, which can be measured in length and width of the tumor. By various methods known in the art, such as, for example, using a caliper for excision from a subject, or using imaging techniques such as bone scan, ultrasound, CT or MRI scan while in the body By measuring the size of the tumor (s).

As used herein, “variable region” or “V region” means a fragment of an IgG chain whose sequence is variable between different antibodies. It extends to Kabat residue 109 in the light chain and 113 in the heavy chain.

The term "buffer" includes agents which maintain a solution pH of the formulation of the invention within an acceptable range, or, in the case of lyophilized formulations of the invention, provide an acceptable solution pH prior to lyophilization.

The terms "freeze-dried," "freeze-dried" and "freeze-dried" refer to the process of first freezing the material to be dried and then removing the ice or frozen solvent by sublimation in a vacuum environment. . Excipients may be included in the pre-freeze dried formulations to enhance the stability of the lyophilized product upon storage.

The term “pharmaceutical formulation” refers to a formulation which is in the form such that the active ingredient is effective and which does not contain additional ingredients which are toxic to the subject to which the agent is to be administered. The terms "formulation" and "pharmaceutical formulation" are used interchangeably throughout.

“Pharmaceutically acceptable” means that excipients (vehicles, additives) and compositions can be reasonably administered to a subject to provide an effective amount of the active ingredient employed, and are considered “generally safe,” eg, physiologically It is tolerated and typically refers to that when administered to a human, does not cause an allergic reaction or an unwanted analogous reaction, such as stomach cramps and the like. In another embodiment, the term is listed for use in animals and more particularly for humans, approved by federal or state regulatory agencies, or listed in US Pharmacopeia or another generally accepted pharmacopeia. Refers to molecular entities and compositions that are present.

A "reconstituted" formulation is one prepared by dissolving a lyophilized protein formulation in a diluent to disperse the protein in the reconstituted formulation. Reconstituted formulations are suitable for administration, such as parenteral administration, and may optionally be suitable for subcutaneous administration.

"Reconstruction time" is the time required to rehydrate the lyophilized formulation into a clear solution free of particles.

A “stable” formulation is one in which the protein inside essentially retains its physical and / or chemical stability and / or biological activity when stored. Various analytical techniques for measuring protein stability are available in the art and are described in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature for a selected time period. For example, in one embodiment, a stable formulation is a formulation in which no significant change is observed for at least 12 months at refrigeration temperature (2-8 ° C.). In another embodiment, the stable formulation is a formulation in which no significant change is observed for at least 18 months at refrigeration temperature (2-8 ° C.). In another embodiment, the stable formulation is a formulation in which no significant change is observed for at least 3 months at room temperature (23-27 ° C.). In another embodiment, the stable formulation is a formulation in which no significant change is observed for at least 6 months at room temperature (23-27 ° C.). In another embodiment, the stable formulation is a formulation in which no significant change is observed for at least 12 months at room temperature (23-27 ° C.). In another embodiment, the stable formulation is a formulation in which no significant change is observed for at least 18 months at room temperature (23-27 ° C.). The criteria for the stability of the antibody formulation are as follows. Typically, up to 10%, preferably 5%, of the antibody monomer is degraded as determined by SEC-HPLC. Typically, the formulation is colorless, or transparent to slightly milky, by visual analysis. Typically, changes in the concentration, pH and osmolality of the formulation do not exceed +/- 10%. Efficacy is typically within 60-140%, preferably 80-120% of the control or reference. Typically, when measured by, for example, HP-SEC, up to 10%, preferably 5%, antibody clipping, ie low molecular weight species (%), is observed. Typically, when measured by, for example, HP-SEC, up to 10%, preferably up to 5% of antibody aggregation, ie high molecular weight species (%), is observed.

When visual inspection of color and / or transparency or as measured by UV light scattering, size exclusion chromatography (SEC) and dynamic light scattering, the antibody does not show a significant increase in aggregation, precipitation and / or denaturation. "Maintaining its physical stability" in the formulation. Changes in protein shape can be assessed by fluorescence spectroscopy to determine protein tertiary structure and FTIR to determine protein secondary structure.

If the antibody did not show significant chemical alteration, then the antibody is "maintaining its chemical stability" in the pharmaceutical formulation. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often alter the chemical structure of proteins include hydrolysis or clipping (eg, assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (eg, peptides with mass spectrometry or MALDI / TOF / MS). Assessed by methods such as by mapping), deamidation (e.g., by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartic acid measurement), and isomerization (isoisopartic acid content determination) And peptide mapping).

If at any given time the biological activity of the antibody is within a predetermined range of biological activity as indicated at the time the pharmaceutical formulation is made, then the antibody is "maintaining its biological activity" in the pharmaceutical formulation. For example, the biological activity of antibodies can be measured by antigen binding assays.

The term "isotonic" means that the osmotic pressure of the agent of interest is essentially the same as human blood. In general, isotonic agents will have an osmotic pressure of about 270-328 mOsm. Slightly hypotonic osmotic pressure is 250-269 mOsm and slightly hypertonic osmotic pressure is 328-350 mOsm. For example, osmotic pressure can be measured using a vapor pressure or ice-freezing type osmotic meter.

II. Formulations of the invention and combination formulations of the invention.

In one aspect, the invention provides a biological agent comprising an anti-TIGIT antibody or antigen binding fragment thereof that specifically binds human TIGIT as an active pharmaceutical ingredient. Incorporation of methionine in the formulation reduces the oxidation of tryptophan in the example of an anti-TIGIT antibody comprising a methionine residue present in the Fc region of the anti-TIGIT antibody, and CDRH3 of SEQ ID NO: 110. The formulation may further comprise a chelator such as DTPA, which may further reduce oxidation.

In one aspect, the invention also provides a combination formulation consisting of anti-TIGIT antibodies in combination with anti-PD-1 antibodies. The major degradation pathways for pembrolizumab included oxidation of methionine 105 (Met105) (eg, M105 of SEQ ID NO: 10) in heavy chain CDR3 upon peroxidase stress, and oxidation of Met105 and Fc methionine residues upon light exposure. Pembrolizumab retained its bioactivity under most stress conditions on the level of degradation tested. However, surface plasmon resonance (SPR) showed a decrease in affinity for PD-1 for peroxidase stressed samples. Exposed methionine residues or methionine residues in the CDRs of the antibody have the potential to affect the biological activity of the antibody through oxidation. Methionine addition can reduce the oxidation of Met105 in the pembrolizumab heavy chain CDRs.

Anti-PD-1 antibodies and antigen binding fragments thereof

In one aspect, the invention provides an anti-human PD-1 antibody or antigen binding fragment thereof (e.g., human or humanized anti-PD-1) that specifically binds human PD-1 as an active pharmaceutical ingredient (PD-1 API) Antibodies and co-formulated anti-TIGIT antibodies or antigen binding fragments thereof, as well as methods of using the agents of the invention. Any anti-PD-1 antibody or antigen binding fragment thereof may be used in the complex formulations and methods of the present invention. In certain embodiments, the PD-1 API is an anti-PD-1 antibody selected from pembrolizumab and nivolumab. In a specific embodiment, the anti-PD-1 antibody is pembrolizumab. In alternative embodiments, the anti-PD-1 antibody is nivolumab. Table 2 below provides the amino acid sequences for the exemplary anti-human PD-1 antibodies, pembrolizumab and nivolumab. Alternative PD-1 antibodies and antigen binding fragments useful in the combination formulations and methods of the present invention are shown in Table 3 below.

In some embodiments, an anti-human PD-1 antibody or antigen binding fragment thereof for use in the complex formulations of the invention comprises three light chain CDRs consisting of CDRL1, CDRL2 and CDRL3 and / or three consisting of CDRH1, CDRH2 and CDRH3 Heavy chain CDRs.

In one embodiment of the invention, CDRL1 is a variant of SEQ ID NO: 1 or SEQ ID NO: 1, CDRL2 is a variant of SEQ ID NO: 2 or SEQ ID NO: 2, CDRL3 of SEQ ID NO: 3 or SEQ ID NO: It is a variant.

In one embodiment, CDRH1 is a variant of SEQ ID NO: 6 or SEQ ID NO: 6, CDRH2 is a variant of SEQ ID NO: 7 or SEQ ID NO: 7, and CDRH3 is a variant of SEQ ID NO: 8 or SEQ ID NO: 8.

In one embodiment, the three light chain CDRs are SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and the three heavy chain CDRs are SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

In an alternative embodiment of the invention, CDRL1 is a variant of SEQ ID NO: 11 or SEQ ID NO: 11, CDRL2 is a variant of SEQ ID NO: 12 or SEQ ID NO: 12, CDRL3 is SEQ ID NO: 13 or SEQ ID NO: 13 Is a variant of.

In one embodiment, CDRH1 is a variant of SEQ ID NO: 16 or SEQ ID NO: 16, CDRH2 is a variant of SEQ ID NO: 17 or SEQ ID NO: 17, and CDRH3 is a variant of SEQ ID NO: 18 or SEQ ID NO: 18.

In one embodiment, the three light chain CDRs are SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and the three heavy chain CDRs are SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

In alternative embodiments, the three light chain CDRs are SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, and the three heavy chain CDRs are SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.

In a further embodiment of the invention, CDRL1 is a variant of SEQ ID NO: 21 or SEQ ID NO: 21, CDRL2 is a variant of SEQ ID NO: 22 or SEQ ID NO: 22, CDRL3 of SEQ ID NO: 23 or SEQ ID NO: 23 It is a variant.

In yet another embodiment, CDRH1 is variant of SEQ ID NO: 24 or SEQ ID NO: 24, CDRH2 is variant of SEQ ID NO: 25 or SEQ ID NO: 25, and CDRH3 of SEQ ID NO: 26 or SEQ ID NO: 26 It is a variant.

In another embodiment, the three light chain CDRs are SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23, and the three heavy chain CDRs are SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26.

Some anti-human PD-1 antibodies and antigen binding fragments of the invention comprise a light chain variable region and a heavy chain variable region. In some embodiments, the light chain variable region comprises a variant of SEQ ID NO: 4 or SEQ ID NO: 4 and the heavy chain variable region comprises a variant of SEQ ID NO: 9 or SEQ ID NO: 9. In further embodiments, the light chain variable region comprises a variant of SEQ ID NO: 14 or SEQ ID NO: 14 and the heavy chain variable region comprises a variant of SEQ ID NO: 19 or SEQ ID NO: 19. In further embodiments, the heavy chain variable region comprises a variant of SEQ ID NO: 27 or SEQ ID NO: 27, and the light chain variable region is of a variant of SEQ ID NO: 28 or SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 29 Variants, or variants of SEQ ID NO: 30 or SEQ ID NO: 30. In this embodiment, the variant light or heavy chain variable region sequence is identical to the reference sequence except for one, two, three, four, or five amino acid substitutions. In some embodiments, substitutions are located within the framework region (ie, outside the CDRs). In some embodiments, one, two, three, four, or five amino acid substitutions are conservative substitutions.

In one embodiment of the combination preparation of the invention, the anti-human PD-1 antibody or antigen binding fragment comprises a light chain variable region comprising or consists of SEQ ID NO: 4 and a heavy chain comprising or consisting of SEQ ID NO: 9 It includes a variable region. In further embodiments, the anti-human PD-1 antibody or antigen binding fragment comprises a light chain variable region comprising or consisting of SEQ ID NO: 14 and a heavy chain variable region comprising or consisting of SEQ ID NO: 19. In one embodiment of the formulation of the invention, the anti-human PD-1 antibody or antigen binding fragment comprises a light chain variable region comprising or consists of SEQ ID NO: 28 and a heavy chain variable comprising or consisting of SEQ ID NO: 27 It includes an area. In a further embodiment, the anti-human PD-1 antibody or antigen binding fragment comprises a light chain variable region comprising or consisting of SEQ ID NO: 29 and a heavy chain variable region comprising or consisting of SEQ ID NO: 27. In another embodiment, the antibody or antigen-binding fragment comprises a light chain variable region comprising or consisting of SEQ ID NO: 30 and a heavy chain variable region comprising or consisting of SEQ ID NO: 27.

In another embodiment, the combination formulation of the present invention is at least 95%, 90%, 85%, 80%, 75% or at least one of an anti-human PD-1 antibody, or one of the V _L domains or V _H domains described above, or V _L domain with 50% sequence homology and / or Antigen-binding proteins having a V _H domain and exhibiting specific binding to PD-1. In another embodiment, the anti-human PD-1 antibody or antigen binding protein of the complex formulation of the invention has a V _L and V _H domain with up to 1, 2, 3, 4, or 5 or more amino acid substitutions. It includes, and shows a specific binding to PD-1.

In any of the above embodiments, the PD-1 API can be a full length anti-PD-1 antibody or antigen binding fragment thereof that specifically binds human PD-1. In certain embodiments, the PD-1 API is a full length anti-PD-1 antibody selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE. Preferably, the antibody is an IgG antibody. Any isotype of IgG can be used, including IgG ₁ , IgG ₂ , IgG ₃ , and IgG ₄ . Different constant domains can be attached to the V _L and V _H regions provided herein. For example, if a particular intended use of an antibody (or fragment) of the invention requires altered effector function, heavy chain constant domains other than IgG1 can be used. Although IgG1 antibodies provide long half-lives and effector functions such as, for example, complement activation and antibody-dependent cellular cytotoxicity, such activity may be undesirable for all uses of the antibody. In this case, for example, an IgG4 constant domain can be used.

In an embodiment of the invention, the PD-1 API comprises a light chain comprising or consisting of the sequence of amino acid residues set forth in SEQ ID NO: 5, and a heavy chain comprising or consisting of the sequence of amino acid residues set forth in SEQ ID NO: 10 It is an anti-PD-1 antibody comprising a. In an alternative embodiment, the PD-1 API comprises a light chain comprising or consisting of the sequence of amino acid residues set forth in SEQ ID NO: 15, and a heavy chain comprising or consisting of the sequence of amino acid residues set forth in SEQ ID NO: 20 It is an anti-PD-1 antibody. In further embodiments, the PD-1 API comprises a light chain comprising or consisting of the sequence of amino acid residues set forth in SEQ ID NO: 32, and a heavy chain comprising or consisting of the sequence of amino acid residues set forth in SEQ ID NO: 31 Is an anti-PD-1 antibody. In further embodiments, the PD-1 API comprises a light chain comprising or consisting of the sequence of amino acid residues set forth in SEQ ID NO: 33, and a heavy chain comprising or consisting of the sequence of amino acid residues set forth in SEQ ID NO: 31 Is an anti-PD-1 antibody. In yet another embodiment, the PD-1 API comprises or consists of a light chain comprising or consisting of the sequence of amino acid residues set forth in SEQ ID NO: 34, and an amino acid residue set forth in SEQ ID NO: 31 Anti-PD-1 antibody comprising a heavy chain. In some combination formulations of the invention, the PD-1 API is pembrolizumab or pembrolizumab biosimilar. In some combination formulations of the invention, the PD-1 API is nivolumab or nivolumab biosimilars.

Usually, amino acid sequence variants of the anti-PD-1 antibodies and antigen binding fragments of the invention, and anti-TIGIT antibodies and antigen binding fragments, comprise the amino acid sequences of the reference antibody or antigen binding fragment (eg heavy chain, light chain, V _H , V _L). , Or humanized sequence) at least 75% amino acid sequence identity, more preferably, at least 80%, more preferably, at least 85%, more preferably, at least 90%, and most preferably, at least 95, 98, or 99 Will have an amino acid sequence with% amino acid sequence identity. Identity or homology with respect to a sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical to the anti-PD-1 residues after aligning the sequences such that the percent sequence identity reaches a maximum and, if necessary, after introducing the gap and Wherein any conservative substitutions are not considered part of the sequence identity. N-terminal, C-terminal, or internal extension, deletion, or insertion into an antibody sequence will not be construed as affecting sequence identity or homology.

Sequence identity refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when the two sequences are optimally aligned. Sequence identity can be measured using the BLAST algorithm, where the parameters of the algorithm are selected such that for each reference sequence of full length, the largest match between each sequence is obtained. The following references relate to the BLAST algorithm frequently used for sequence analysis: BLAST ALGORITHMS: Altschul, SF, et al ., (1990) J. Mol. Biol. 215: 403-410; Gish, W., et al ., (1993) Nature Genet. 3: 266-272; Madden, TL, et al. , (1996) Meth. Enzymol. 266: 131-141; Altschul, SF, et al. , (1997) Nucleic Acids Res. 25: 3389-3402; Zhang, J., et al. , (1997) Genome Res. 7: 649-656; Wootton, JC, et al. , (1993) Comput. Chem. 17: 149-163; Hancock, JM et al. , (1994) Comput. Appl. Biosci. 10: 67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, MO, et al. , "A model of evolutionary change in proteins." in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. MO Dayhoff (ed.), Pp. 345-352, Natl. Biomed. Res. Found., Washington, DC; Schwartz, RM, et al. , "Matrices for detecting distant relationships." in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. "MO Dayhoff (ed.), Pp. 353-358, Natl. Biomed. Res. Found., Washington, DC; Altschul, SF, (1991) J. Mol. Biol. 219: 555-565. States, DJ, et al. , (1991) Methods 3: 66-70; Henikoff, S., et al. , (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919; Altschul, SF, et al. , (1993) J. Mol. Evol. 36: 290-300; ALIGNMENT STATISTICS: Karlin, S., et al. , (1990) Proc. Natl. Acad. Sci. USA 87: 2264-2268; Karlin, S., et al. , (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877; Dembo, A., et al. , (1994) Ann Prob. 22: 2022-2039; and Altschul, SF "Evaluating the statistical significance of multiple distinct local alignments." In Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), (1997) pp. 1 -14, Plenum, New York.

Similarly, light chain classes may also be used in the compositions and methods herein. In particular, kappa, lambda, or variants thereof are useful in the present compositions and methods.

TABLE 2

Exemplary PD-1 Antibody Sequences

TABLE 3

Additional PD-1 antibodies and antigen binding fragments useful in the complex formulations, methods and uses of the present invention.

In some embodiments of the co-formulations of the invention, the PD-1 API (ie, anti-PD-1 antibody or antigen binding fragment thereof) is present at a concentration of about 25 mg / mL to about 100 mg / mL. In alternative embodiments, the API is present at a concentration of about 10 mg / mL, about 25 mg / mL, about 50 mg / mL, about 75 mg / mL, or about 100 mg / mL.

Anti-TIGIT antibodies and antigen binding fragments thereof

In one aspect, the present invention is directed to a biological agent comprising an anti-TIGIT antibody or antigen-binding fragment thereof (eg, a human or humanized anti-TIGIT antibody) that specifically binds human TIGIT as an active pharmaceutical ingredient (TIGIT API), It provides a method of using the formulation of the present invention.

In another aspect, the invention also relates to (i) an anti-TIGIT antibody or antigen binding fragment thereof that specifically binds human TIGIT (eg, a human or humanized anti-TIGIT antibody) and (ii) specific to human PD-1 An anti-human PD-1 antibody or antigen-binding fragment thereof that binds to an agent is provided. Any anti-TIGIT antibody or antigen binding fragment thereof can be used in the formulations and methods, including combination formulations of the invention. Exemplary anti-TIGIT antibody sequences are described in Tables 4 and 5 below.

TABLE 4

Exemplary Anti-TIGIT Antibodies

In some embodiments the anti-TIGIT antibody or antigen binding fragment thereof comprises three light chain CDRs consisting of CDRL1, CDRL2, and CDRL3 and / or three heavy chain CDRs consisting of CDRH1, CDRH2, and CDRH3.

In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof is CDRH1 comprising SEQ ID NO: 35, CDRH2 comprising SEQ ID NO: 36, SEQ ID NO: 37, 103, 104, 105, 106, 107, or 160 CDRH3 comprising any of, CDRL1 comprising SEQ ID NO: 38, CDRL2 comprising any of SEQ ID NO: 39, 89, 90, 91, 92, 93, 94, 95, 96, 97, or 69, And CDRL3 comprising any of SEQ ID NOs: 40, 98, 99, 100, 101, 102, or 162.

In another embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises CDRH1 comprising SEQ ID NO: 81, CDRH2 comprising SEQ ID NO: 82, CDRH3 comprising SEQ ID NO: 83, SEQ ID NO: 84 CDRL1, CDRL2 comprising SEQ ID NO: 85, and CDRL3 comprising SEQ ID NO: 86.

In another embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof is CDRH1 comprising SEQ ID NO: 108, SEQ ID NO: 109, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, CDRH2 comprising any of 126, 127, 128, 129, 130, 131, 154, 155 or 167, SEQ ID NO: 110, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, CDRH3 comprising any of 183, 184, 185, 186 or 187, CDRL1 comprising SEQ ID NO: 111, SEQ ID NO: 112, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 CDRL2 comprising any of, 142 or 168, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 113.

In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof is CDRH1 comprising SEQ ID NO: 35, CDRH2 comprising SEQ ID NO: 36, CDRH3 comprising SEQ ID NO: 37, CDRL1 comprising SEQ ID NO: 38 , CDRL2 comprising SEQ ID NO: 39, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 40.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof is CDRH1 comprising SEQ ID NO: 108, CDRH2 comprising any of SEQ ID NO: 109, 154 or 145, CDRH3 comprising SEQ ID NO: 110, sequence CDRL1 comprising SEQ ID NO: 111, CDRL2 comprising SEQ ID NO: 112, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 113.

In another embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof is CDRH1 comprising the amino acid sequence of SEQ ID NO: 108, CDRH2 comprising SEQ ID NO: 154, CDRH3 comprising SEQ ID NO: 110, SEQ ID NO: 111 CDRL1 comprising, CDRL2 comprising SEQ ID NO: 112, and CDRL3 comprising SEQ ID NO: 113.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a variable heavy chain region and a variable light chain variable region. In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a variable heavy chain region comprising SEQ ID NO: 41 and a variable light chain region comprising SEQ ID NO: 42.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a variable heavy chain region comprising SEQ ID NO: 87, and a variable light chain region comprising SEQ ID NO: 88.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a variable heavy chain region comprising SEQ ID NO: 114 and a variable light chain region comprising SEQ ID NO: 115.

In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a variable heavy chain region comprising any of SEQ ID NOs: 43-58, 65-75, and 87, and SEQ ID NOs: 59-64, 76-80, and 88 A variable light chain region comprising any of the above.

In an embodiment, an anti-TIGIT antibody or antigen binding fragment thereof comprises a variable heavy chain region comprising any of SEQ ID NOs: 144-149 and a variable light chain region comprising any of SEQ ID NOs: 150-153.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a variable heavy chain region comprising SEQ ID NO: 148 and a variable light chain region comprising SEQ ID NO: 152.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a variable heavy chain region comprising SEQ ID NO: 147 and a variable light chain region comprising SEQ ID NO: 150.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a variable heavy chain region comprising SEQ ID NO: 148 and a variable light chain region comprising SEQ ID NO: 153.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a variable heavy chain region comprising SEQ ID NO: 163 and a variable light chain region comprising SEQ ID NO: 165.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a variable heavy chain region comprising SEQ ID NO: 169 and a variable light chain region comprising SEQ ID NO: 171.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a variable heavy chain region comprising SEQ ID NO: 164 and a variable light chain region comprising SEQ ID NO: 166.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a variable heavy chain region comprising SEQ ID NO: 170 and a variable light chain region comprising SEQ ID NO: 172.

TABLE 5

Exemplary Sequences of Anti-TIGIT Antibodies

In one embodiment, the anti-TIGIT antibody or antigen binding fragment is CDRH1 comprising SEQ ID NO: 188, CDRH2 comprising SEQ ID NO: 189, CDRH3 comprising any of SEQ ID NOs: 190, 220, 221, or 222. , CDRL1 comprising SEQ ID NO: 191, CDRL2 comprising SEQ ID NO: 192, and CDRL3 comprising any of SEQ ID NOs: 193, 232, 233, 234, 235, 236, or 237.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises any of CDRH1, SEQ ID NO: 205, 256, 257, 258, 259, 260, 261, 262, or 263, including SEQ ID NO: 204. CDRH2, CDRH3 comprising SEQ ID NO: 206, CDRL1 comprising SEQ ID NO: 207, CDRL2 comprising SEQ ID NO: 208, and CDRL3 comprising SEQ ID NO: 209.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a variable heavy chain region and a variable light chain variable region. In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a variable heavy chain region comprising SEQ ID NO: 194 and a variable light chain region comprising SEQ ID NO: 195.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a variable heavy chain region comprising SEQ ID NO: 196 and a variable light chain region comprising SEQ ID NO: 200.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a variable heavy chain region comprising SEQ ID NO: 210 and a variable light chain region comprising SEQ ID NO: 211.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a variable heavy chain region comprising SEQ ID NO: 212 and a variable light chain region comprising SEQ ID NO: 216.

In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof has a variable heavy chain comprising any of SEQ ID NOs: 197, 198, 199, 223, 224, 225, 226, 227, 228, 229, 230, and 231. Region, and any of SEQ ID NOs: 201, 202, 203, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, and 255 It includes a variable light chain region including that of.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof is SEQ ID NOs: 213, 214, 215, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, A variable heavy chain region comprising any of 277, 278, 279, 280, 281, 282, 283, 284, 285, and 286, and a variable light chain region comprising any of SEQ ID NOs: 217, 218, and 219 Include.

Additional anti-TIGIT antibodies that can be used in the formulations described herein are described, for example, in PCT International Application No. WO 2016/106302; WO 2016/011264; And those disclosed in WO 2009/126688.

TABLE 6

Exemplary Heavy Chain Sequences

In any of the aforementioned embodiments, the anti-TIGIT antibody or antigen binding fragment thereof is an antibody comprising any of the variable heavy chains described above and any human heavy chain constant domains. In one embodiment, the antibody or antigen-binding fragment thereof of the invention is of the IgG isotype and comprises human IgG1, IgG2, IgG3 or IgG4 human heavy chain constant domains. In one embodiment, an antibody or antigen-binding fragment thereof of the invention comprises a human heavy chain IgG1 constant domain (SEQ ID NO: 291) or a variant thereof, wherein the variant is one comprising up to 20 modified amino acid substitutions. In one embodiment, the antibody or antigen-binding fragment thereof of the invention comprises a human heavy chain IgG1 constant domain comprising the amino acid sequence of SEQ ID NO: 291. In one embodiment, the antibody or antigen-binding fragment thereof of the invention comprises a human heavy chain IgG1 constant domain, wherein the IgG1 constant domain is afucosylated. In one embodiment, an antibody or antigen-binding fragment thereof of the invention comprises a human heavy chain IgG4 constant domain or variant thereof, wherein the variant is one comprising up to 20 modified amino acid substitutions. In another embodiment, an antibody or antigen-binding fragment thereof of the invention comprises a human heavy chain IgG4 constant domain, wherein the amino acid at position 228 is substituted for Ser at Pro (using the EU numbering system). In one embodiment, the antibody or antigen-binding fragment thereof of the invention comprises a human heavy chain IgG4 constant domain comprising the amino acid sequence of SEQ ID NO: 292.

In any of the aforementioned embodiments, the anti-TIGIT antibody or antigen binding fragment thereof may comprise any of the variable light chains described above and the human light chain constant domains. In one embodiment, an antibody or antigen-binding fragment thereof of the invention comprises a human kappa light chain constant domain or variant thereof, wherein the variant is one comprising up to 20 modified amino acid substitutions. In another embodiment, an antibody or antigen-binding fragment thereof of the invention comprises a human lambda light chain constant domain or variant thereof, wherein the variant is one comprising up to 20 modified amino acid substitutions. In one embodiment, the antibody or antigen-binding fragment thereof of the invention comprises a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO: 293.

Formulation

The formulations of the present invention minimize the formation of antibody aggregates (high molecular weight species) and particulate, high molecular weight and low molecular weight species, minimize oxidation of methionine residues, and enable the antibody to reliably retain its biological activity over time. do.

In one aspect, the invention includes various agents of anti-TIGIT antibodies, or antigen binding fragments thereof. For example, the present invention provides a kit comprising (i) an anti-TIGIT antibody or antigen binding fragment thereof, (ii) a buffer (eg L-histidine or acetate), (iii) a non-reducing sugar (eg sucrose); (iv) nonionic surfactants (eg, polysorbate 80); And (v) antioxidants (eg, L-methionine). In one embodiment, the formulation further comprises an anti-PD1 antibody. In one embodiment, the formulation further comprises a chelator. In one embodiment, the chelator is present in an amount from about 1 μM to about 50 μM. In one embodiment, the chelator is diethylenetriaminepentaacetic acid (DTPA). In another embodiment, the chelator is EDTA.

In another aspect, the invention also encompasses various complex preparations of anti-TIGIT antibodies, or antigen binding fragments thereof and anti-human PD-1 antibodies, or antigen binding fragments thereof. In one embodiment, the formulation comprises (i) an anti-TIGIT antibody, or antigen binding fragment thereof, (ii) an anti-human PD-1 antibody or antigen binding fragment thereof, (iii) a buffer (eg, L-histidine or acetate) , (iv) non-reducing sugars (eg sucrose), (v) nonionic surfactants (eg polysorbate 80), and (vi) antioxidants (eg L-methionine). In one embodiment, the formulation further comprises a chelator. In one embodiment, the chelator is present in an amount from about 1 μM to about 50 μM. In one embodiment, the chelator is diethylenetriaminepentaacetic acid (DTPA). In another embodiment, the chelator is EDTA.

Pharmaceutical formulations of the present invention may comprise a buffer.

Useful buffers in the pharmaceutical formulations and methods of the present invention include succinate (sodium succinate or potassium succinate), L-histidine, phosphate (sodium phosphate or potassium phosphate), tris (tris (hydroxymethyl) aminomethane), diethanolamine , Citrate (sodium citrate), acetate (sodium acetate) and the like. In one embodiment of the invention, the buffer is about 1-20 mM (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 mM). In specific embodiments of the invention, the buffer is a histidine, such as L-histidine buffer.

The pH of the buffer of the invention is about 4.5 to about 6.5; About 5.0-6.2; In the range of about 5.5-6.0; Preferably, the pH is about 5.8. Upon reaching an exemplary formulation, histidine and acetate buffers in the pH range 5.0-6.0 were studied for suitability. For example, when a pH value is referred to as a range, such as "pH is pH 5.5 to 6.0," the range is intended to include the stated value. For example, the range of about 5.0 to about 6.0 includes 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, and 6.0. Unless otherwise specified, pH refers to the pH after reconstitution of the lyophilized formulation of the invention. pH is typically measured at 25 ° C. using a standard glass bulb pH meter. As used herein, a solution comprising “histidine buffer of pH X” is pH X and refers to a solution comprising histidine buffer, ie, pH is intended to refer to the pH of the solution.

In one embodiment of the invention, in one embodiment of the invention, the anti-TIGIT agent and the complex agent consisting of anti-TIGIT and anti-human PD-1 comprise a non-reducing sugar. As used herein, a "non-reducing sugar" is a sugar that does not contain free aldehyde groups or free ketone groups or cannot act as a reducing agent because it cannot be converted to contain them. Examples of non-reducing sugars include, but are not limited to, disaccharides such as sucrose and trehalose. In one embodiment of the invention, the non-reducing sugar is about 1-10% (w / v) (1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% (w / v)) Present in quantities. In another embodiment, the non-reducing sugar is present in an amount of about 6% (w / v) to about 8% (w / v) (6, 7, or 8% (w / v)). In further embodiments, the non-reducing sugar is present in an amount of about 6% (w / v). In further embodiments, the non-reducing sugar is present in an amount of about 7% (w / v). In further embodiments, the non-reducing sugar is present in an amount of about 8% (w / v). In one embodiment, non-reducing sugar sucrose, trehalose, or raffinose. In another embodiment, the non-reducing sugar is sucrose. In further embodiments, sucrose is present at 6-8% w / v. In one embodiment, sucrose is present at about 6% (w / v). In one embodiment, sucrose is present at about 7% (w / v). In one embodiment, sucrose is present at about 8% (w / v).

Formulations of the present invention also include surfactants. As used herein, a surfactant is a surface active agent that is substantially amphoteric. Surfactants may be used to provide stability, to reduce and / or prevent aggregation, or to prevent and / or inhibit protein damage during processing conditions, such as during purification, filtration, freeze drying, transport, storage and delivery. May be added to the formulations herein. In the present invention, surfactants may be useful for providing additional stability to the active ingredient (s).

Nonionic surfactants that may be useful in formulations, including complex formulations of the invention, include polysorbate-20 (polyoxyethylene sorbitan monolaurate), polysorbate-40 (polyoxyethylene sorbitan monopalmitate). Polyoxyethylene sorbitan fatty acid ester (Tween®), including polysorbate-60 (polyoxyethylene sorbitan monostearate), and polysorbate-80 (polyoxyethylene sorbitan monooleate) (Polysorbate, sold under the trade name Uniquema Americas LLC, Wilmington, Delaware, USA); Polyoxyethylene alkyl ethers such as Brij® 58 (Unichema America LLC: Wilmington, Delaware, USA) and Brij® 35; Poloxamers (eg, poloxamer 188); Triton® X-100 (Union Carbide Corp., Houston, Tex.) And Triton® X-114; NP40; Span 20, span 40, span 60, span 65, span 80 and span 85; Copolymers of ethylene and propylene glycol (eg, Pluronic® series of nonionic surfactants such as Pluronic® F68, Pluronic® 10R5, Pluronic® F108, Pluronic® F127, Pluronic® F38, Pluronic® L44, Pluronic® L62 (BASF Corp .: Ludwigshafen, Ludwigshafen, Germany); and Sodium Dodecyl Sulfate (SDS) In one embodiment, the nonionic surfactant is polysorbate 80 or polysorbate 20. In one embodiment, the nonionic surfactant is polysorbate 20. In another embodiment, the nonionic surfactant is poly Sorbate 80.

The amount of nonionic surfactant included in the formulation of the present invention is an amount sufficient to perform a desired function, i.e., an active pharmaceutical ingredient (ie, an anti-TIGIT antibody or antigen-binding fragment thereof, or an anti-TIGIT antibody or antigen thereof) in the formulation. Binding fragment and anti-human PD-1 antibody or antigen binding fragment thereof, both). All percentages listed for nonionic surfactants are listed in w / v%. Typically, the surfactant is present at a concentration of about 0.008% w / v to about 0.1% w / v. In some embodiments of this aspect of the invention, the surfactant is about 0.01% to about 0.1% in the formulation; About 0.01% to about 0.09%; About 0.01% to about 0.08%; About 0.01% to about 0.07%; About 0.01% to about 0.06%; About 0.01% to about 0.05%; About 0.01% to about 0.04%; About 0.01% to about 0.03%, about 0.01% to about 0.02%, about 0.015% to about 0.04%; Present in an amount of about 0.015% to about 0.03%, about 0.015% to about 0.02%, about 0.02% to about 0.04%, about 0.02% to about 0.035%, or about 0.02% to about 0.03%. In specific embodiments, the surfactant is present in an amount of about 0.02%. In alternative embodiments, the surfactant is present in an amount of about 0.01%, about 0.015%, about 0.025%, about 0.03%, about 0.035%, or about 0.04%.

In an exemplary embodiment of the invention, the surfactant is a nonionic surfactant selected from the group consisting of polysorbate 20 and polysorbate 80. In a preferred embodiment, the surfactant is polysorbate 80.

In a specific embodiment, the formulations, including the composite formulations of the present invention, comprise about 0.01% w / v to about 0.04% w / v polysorbate 80. In a further embodiment, the formulations described herein comprise polysorbate 80 in an amount of about 0.008% w / v, about 0.01% w / v. In one embodiment, the amount of polysorbate 80 is about 0.015 w / v%. In another embodiment, the amount of polysorbate 80 is about 0.02% w / v. In further embodiments, the amount of polysorbate 80 is about 0.025% w / v. In another embodiment, the amount of polysorbate 80 is about 0.03% w / v. In further embodiments, the amount of polysorbate 80 is about 0.035% w / v. In another embodiment, the amount of polysorbate 80 is about 0.04% w / v. In further embodiments, the amount of polysorbate 80 is about 0.045% w / v. In certain embodiments, the formulations of the invention comprise about 0.02% w / v polysorbate 80.

Formulations, including complex formulations of the invention, also include methionine, or a pharmaceutically acceptable salt thereof, as an antioxidant. In one embodiment, the methionine is L-methionine. In another embodiment, methionine is a pharmaceutically acceptable salt of L-methionine, such as, for example, methionine HCl. In one embodiment of the invention, the methionine is in the formulation about 1-20 mM (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 mM). In another embodiment, the methionine is present from about 5 mM to about 10 mM (5, 6, 7, 8, 9 and 10 mM). In another embodiment, methionine is present at about 10 mM.

Formulations, including complex formulations of the invention, may also further comprise chelating agents. In one embodiment of the invention, the chelating agent is present in the formulation at a concentration of about 5-30 μM (eg, 5, 10, 15, 20, 25, or 30 μM). In one embodiment, the chelating agent is DTPA. In another embodiment, the chelating agent is EDTA.

Lyophilized Pharmaceutical Composition

Lyophilized formulations of therapeutic proteins provide several advantages. Lyophilized formulations generally provide better chemical stability than solution formulations and therefore provide increased half-life. Lyophilized formulations can also be reconstituted at different concentrations depending on clinical factors such as administration or route of administration. For example, lyophilized formulations can be reconstituted at high concentrations (ie, in small volumes) if necessary for subcutaneous administration, or at lower concentrations when administered intravenously. If a high dose of dosage is required for a particular subject, high concentrations may also be required, especially if subcutaneously administered if the injection volume should be minimized. One such lyophilized antibody formulation is disclosed in US Pat. No. 6,267,958, which is incorporated herein by reference in its entirety. Another lyophilized formulation of the therapeutic protein is disclosed in US Pat. No. 7,247,707, which is incorporated herein by reference in its entirety.

Typically, lyophilized formulations expect reconstitution of high concentrations of drug product (DP, in an exemplary embodiment, humanized anti-PD-1 antibody pembrolizumab, or antigen binding fragment thereof), ie, low volume of water. Expected to be reconstituted and prepared. Subsequently, dilution with water or isotonic buffer can then readily be used to dilute DP to lower concentrations. Typically, excipients are included in the lyophilized formulations of the present invention to a level that will result in an almost isotonic formulation, for example when reconstituted at high DP concentrations for subcutaneous administration. Reconstitution in a larger volume of water to provide a lower DP concentration will inevitably reduce the tonicity of the reconstituted liquid, but such reduction may be of little importance for non-subcutaneous administration, such as intravenous administration. If isotonicity is desired at lower DP concentrations, the lyophilized powder may be reconstituted in a standard low volume of water and then further diluted with an isotonic diluent such as 0.9% sodium chloride.

Lyophilized formulations of the present invention are formed by lyophilization (freeze-drying) of pre-freeze-dried solutions. Freeze-drying is accomplished by freezing the formulation and then subliming the water at a temperature suitable for primary drying. Under these conditions, the product temperature is below the eutectic point or collapse temperature of the formulation. Typically, the shelf temperature for the primary drying will be in the range of about -30 to 25 ° C. at a suitable pressure, typically at a pressure ranging from about 50 to 250 mTorr, provided the product remains frozen during the primary drying. . The size and type of formulation, container (eg, glass vial) holding the sample, and volume of liquid will depend on the time required for drying, which may range from several hours to several days (eg, 40-60 hr). The secondary drying step can be carried out at about 0-40 ° C., depending primarily on the type of protein used and the type and size of the container. Secondary drying time depends on the desired residual moisture level in the product, and typically takes at least about 5 hours. Typically, the water content of the lyophilized formulation is less than about 5%, preferably less than about 3%. The pressure may be the same as used during the first drying step. Freeze-drying conditions may vary depending on formulation and vial size.

In some cases, it may be desirable to lyophilize the protein preparation in a container in which reconstitution of the protein is performed to avoid the transfer step. In such a case, the container may be, for example, 3, 5, 10, 20, 50 or 100 cc vials.

Lyophilized formulations of the invention are reconstituted prior to administration. The protein may have a concentration of about 10, 15, 20, 25, 30, 40, 50, 60, 75, 80, 90, or more than 100 mg / mL, such as 150 mg / mL, 200 mg / mL, 250 mg / mL, or at a concentration of 300 mg / mL up to about 500 mg / mL. In one embodiment, the protein concentration after reconstitution is about 10-300 mg / ml. In one embodiment, the protein concentration after reconstitution is about 20-250 mg / ml. In one embodiment, the protein concentration after reconstitution is about 150-250 mg / ml. In one embodiment, the protein concentration after reconstitution is about 180-220 mg / ml. In one embodiment, the protein concentration after reconstitution is about 50-150 mg / ml. In one embodiment, the protein concentration after reconstitution is about 100 mg / ml. In one embodiment, the protein concentration after reconstitution is about 75 mg / ml. In one embodiment, the protein concentration after reconstitution is about 50 mg / ml. In one embodiment, the protein concentration after reconstitution is about 25 mg / ml. High protein concentrations are particularly useful when subcutaneous delivery of reconstituted agents is intended. However, for other routes of administration, such as intravenous administration, lower protein concentrations may be desirable (eg, about 5-50 mg / mL).

Reconstitution is generally carried out at a temperature of about 25 ° C. to ensure that it can be fully hydrated, although other temperatures may be used if desired. The time required for reconstitution will depend, for example, on the type of diluent, amount of excipient (s) and protein. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (eg, phosphate buffered solutions), sterile saline, Ringer's solution, or dextrose solution.

Liquid pharmaceutical composition

Liquid antibody preparations can be prepared by taking the drug substance in liquid form (eg, anti-humanized PD-1) (eg, pembrolizumab in an aqueous pharmaceutical formulation) and exchanging its buffer with the desired buffer as the last step of the purification process. have. There is no lyophilization step in this embodiment. The drug substance in the final buffer is concentrated to the desired concentration. Excipients such as sucrose and polysorbate 80 are added to the drug substance and diluted to the final protein concentration using an appropriate buffer. The formulated final drug substance is filtered using a 0.22 μm filter and filled into a final container (eg glass vial).

III. How to use

The invention also provides an effective amount of any of the formulations of the invention to a subject; That is, to a method of treating cancer in a subject, comprising administering any of the agents described herein. In some specific embodiments of the method, the formulation is administered to the subject via intravenous administration. In other embodiments, the formulation is administered to the subject via subcutaneous administration. In one embodiment, the invention includes a method of treating cancer in a human patient comprising administering any agent of the invention to a human patient.

In any of the methods of the invention, the cancer is melanoma, lung cancer, head and neck cancer, bladder cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular cancer, lymphoma, renal cancer, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary cancer, colon To be selected from the group consisting of rectal cancer, cervical cancer, thyroid cancer, salivary gland cancer, prostate cancer (eg, hormone refractory prostate adenocarcinoma), pancreatic cancer, colon cancer, esophageal cancer, liver cancer, thyroid cancer, glioblastoma, glioma, and other neoplastic malignancies. Can be.

In some embodiments, the lung cancer is non-small cell lung cancer.

In alternative embodiments, the lung cancer is small cell lung cancer.

In some embodiments, the lymphoma is Hodgkin's lymphoma.

In other embodiments, the lymphoma is non-Hodgkin's lymphoma. In certain embodiments, the lymphoma is mediastinal large B cell lymphoma.

In some embodiments, the breast cancer is triple negative breast cancer.

In further embodiments, the breast cancer is ER + / HER2- breast cancer.

In some embodiments, the bladder cancer is urinary tract cancer.

In some embodiments, the head and neck cancer is nasopharyngeal cancer. In some embodiments, the cancer is thyroid cancer. In other embodiments, the cancer is salivary gland cancer. In other embodiments, the cancer is head and neck squamous cell carcinoma.

In one embodiment, the present invention includes a method of treating metastatic non-small cell lung cancer (NSCLC) in a human patient comprising administering an agent of the invention to a human patient. In a specific embodiment, the patient is suffering from a tumor with high PD-L1 expression rate ((tumor ratio score (TPS) ≧ 50%)] and has never been previously treated with platinum containing chemotherapy. In another embodiment, the patient is suffering from a tumor showing PD-L1 expression (TPS ≧ 1%) and has been previously treated with platinum containing chemotherapy. In another embodiment, the patient is suffering from a tumor showing PD-L1 expression (TPS ≧ 1%) and has never been previously treated with platinum containing chemotherapy. In specific embodiments, the patient is a patient who has developed disease progression after or after receiving platinum containing chemotherapy. In certain embodiments, PD-L1 TPS is measured by FDA approval test. In certain embodiments, the patient's tumor is one that exhibits no EGFR or ALK genome abnormalities. In certain embodiments, the patient's tumor exhibits an EGFR or ALK genome abnormality and, prior to receiving an anti-PD-1 antibody, or antigen binding fragment thereof, when treated for EGFR or ALK abnormality (s), or thereafter. The patient showed progress in the disease.

In some embodiments, the cancer is high level of microsatellite instability (MSI-H) of metastatic colorectal cancer.

In some embodiments, the cancer is high level of microsatellite instability (MSI-H) of metastatic colorectal cancer.

In some embodiments, the cancer is a solid tumor of high levels of microsatellite instability (MSI-H).

In some embodiments, the cancer is a solid tumor with a high mutation burden.

In some embodiments, the cancer is selected from the group consisting of melanoma, non-small cell lung cancer, recurrent or refractory classical Hodgkin's lymphoma, head and neck squamous cell carcinoma, urinary tract cancer, esophageal cancer, gastric cancer, and hepatocellular carcinoma.

In another embodiment of the method of treatment, the cancer is hematological cancer. In certain embodiments, the hematologic cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large B cell lymphoma (DLBCL), EBV- Benign DLBCL, Primary Mediastinal Large B Cell Lymphoma, T Cell / Histomeric Superior Large B Cell Lymphoma, Vesicular Lymphoma, Hodgkin's Lymphoma (HL), Mantle Cell Lymphoma (MCL), Multiple Myeloma (MM), Myeloid Cell Leukemia-1 Protein (Mcl-1), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), or small lymphocytic lymphoma (SLL).

Malignant tumors showing improved disease-free survival and overall survival with respect to the presence of tumor-infiltrating lymphocytes in biopsy or surgical material, such as melanoma, colorectal cancer, liver cancer, kidney cancer, gastric / esophageal cancer, breast cancer, pancreatic cancer and ovarian cancer It is included in the methods and treatments described herein. Such cancer subtypes are known to be sensitive to immune control by T lymphocytes. In addition, refractory or recurrent malignancies may be included in which growth can be inhibited using the antibodies described herein.

Additional cancers that may benefit from treatment with the agents described herein include, for example, causally related to Kaposi's sarcoma, liver cancer, nasopharyngeal cancer, lymphoma, cervical cancer, vulvar cancer, anal cancer, penile cancer, and oral cancer. And those associated with persistent infection with viruses such as human immunodeficiency virus known to be known, hepatitis virus classes A, B and C, Epstein Barr virus, human papilloma virus.

The formulations may also be used to prevent or treat infections and infectious diseases. Accordingly, the present invention provides a method of treating a chronic infection in a mammalian subject comprising administering to the mammalian subject an effective amount of an agent of the invention. In some specific embodiments of the method, the formulation is administered to the subject via intravenous administration. In other embodiments, the formulation is administered to the subject by subcutaneous administration.

Such agents may be used alone or in combination with vaccines to stimulate an immune response to pathogens, toxins and self-antigens. Antibodies or antigen-binding fragments thereof are infectious to humans, including but not limited to human immunodeficiency virus, hepatitis virus classes A, B and C, Epstein Barr virus, human cytomegalovirus, human papilloma virus, and herpes virus. It can be used to stimulate an immune response to the virus. Antagonist anti-PD-1 antibodies or antibody fragments can be used to stimulate an immune response to infection with bacteria or fungal parasites, and other pathogens. Hepatitis B and C and viral infections with HIV are considered to be particularly chronic viral infections.

The formulations of the invention can be administered to a patient in combination with one or more "additional therapeutic agents." Additional therapeutic agents include, but are not limited to, antibodies to biotherapeutics (VEGF, EGFR, Her2 / neu, VEGF receptors, other growth factor receptors, CD20, CD40, CD-40L, OX-40, 4-1BB, and ICOS). Immunogenic agents (eg, attenuated cancerous cells, tumor antigens, antigen presenting cells such as dendritic cells pulsed with tumor-derived antigens or nucleic acids, immune stimulating cytokines (eg, IL-2, IFNα2) , GM-CSF), and, for example, but not limited to, cells transfected with a gene encoding an immune stimulating cytokine such as GM-CSF).

As mentioned above, in some embodiments of the methods of the invention, the method further comprises administering an additional therapeutic agent. In certain embodiments, the additional therapeutic agent is an anti-LAG3 antibody or antigen binding fragment thereof, anti-GITR antibody, or antigen binding fragment thereof, anti-CTL4 antibody, or antigen binding fragment thereof, anti-CD27 antibody or antigen binding fragment thereof. . In one embodiment, the additional therapeutic agent is a Newcastle Disease Virus vector expressing IL-12. In further embodiments, the additional therapeutic agent is dinaciclip. In yet further embodiments, the additional therapeutic agent is a STING agonist.

Suitable routes of administration include, for example, parenteral delivery, including intramuscular, subcutaneous, as well as intradural, direct intraventricular, intravenous, intraperitoneal delivery. The drug can be administered in a variety of conventional ways, such as by intraperitoneal, parenteral, intraarterial or intravenous injection. For modes of administration (eg, subcutaneous administration) where the volume of the solution should be limited, the lyophilized formulation should be able to be reconstituted at high concentrations.

Selecting a dosage of the additional therapeutic agent depends on several factors, including the serum or tissue replacement rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells, tissues or organs within the subject being treated. The dosage of the additional therapeutic agent should be an amount that provides an acceptable level of side effects. Thus, the dosage and frequency of administration of each additional therapeutic agent (eg, biotherapeutic or chemotherapeutic agent) will depend in part on the particular therapeutic agent, severity of the cancer being treated, and patient characteristics. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules is available. See, eg, Wawrzynczak (1996) Antibody Therapy , Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis , Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases , Marcel Dekker, New York, NY; Baert et al . (2003) New Engl . J. Med . 348: 601-608; Milgrom et al . (1999) New Engl. J. Med . 341: 1966-1973; Slamon et al . (2001) New Engl . J. Med . 344: 783-792; Benaminovitz et al . (2000) New Engl . J. Med . 342: 613-619; Ghosh et al . (2003) New Engl . J. Med . 348: 24-32; Lipsky et al . (2000) New Engl. J. Med . 343: 1594-1602; Physicians 'Desk Reference 2003 (Physicians' Desk Reference, 57th Ed); Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002). Appropriate dosage regimens can be determined by a clinician, for example, using parameters or factors known in the art to be affecting the treatment, suspected, or predicted to affect the treatment, eg For example, it will depend on the clinical history of the patient (eg, existing therapy), the type and stage of cancer to be treated, and the biomarker of the response to one or more of the therapeutic agents in the combination therapy.

Various references are available to facilitate the selection of pharmaceutically acceptable carriers or excipients for further therapeutic agents. See, eg, Remington's Pharmaceutical Sciences and US . Pharmacopeia: National Formulary , Mack Publishing Company, Easton, PA (1984); Hardman et al . (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics , McGraw-Hill, New York, NY; Gennaro (2000) Remington : The Science and Practice of Pharmacy , Lippincott, Williams, and Wilkins, New York, NY; Avis et al . (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications , Marcel Dekker, NY; Lieberman, et al . (eds.) (1990) Pharmaceutical Dosage Forms: Tablets , Marcel Dekker, NY; Lieberman et al . (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems , Marcel Dekker, NY; See Weiner and Kotkoskie (2000) Excipient Toxicity and Safety , Marcel Dekker, Inc., New York, NY.

The pharmaceutical antibody preparations can be administered by continuous infusion or at intervals of, for example, 1 day, 1-7 times per week, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, and the like. Preferred dosing protocols involve a maximum dose or dosing frequency that avoids significant undesirable side effects. Total weekly doses are generally at least 0.05 μg / kg (body weight), 0.2 μg / kg (body weight), 0.5 μg / kg (body weight), 1 μg / kg (body weight), 10 μg / kg (body weight), 100 μg / kg (body weight), 0.2 mg / kg (body weight), 1.0 mg / kg (body weight), 2.0 mg / kg (body weight), 10 mg / kg (body weight), 25 mg / kg (body weight), 50 mg / kg (Weight) or more. See, eg, Yang et al. (2003) New Engl . J. Med . 349: 427-434; Herold et al. (2002) New Engl . J. Med. 346: 1692-1698; Liu et al. (1999) J. Neurol . Neurosurg . Psych. 67: 451-456; Portielji et al . (20003) Cancer Immunol . Immunother . 52: 133-144. Desired doses of small molecule therapeutics, such as peptide mimetics, natural products, or organic chemicals, are approximately the same as for antibodies or polypeptides on a molar / kg basis.

Embodiments of the invention also include (a) therapy (eg, therapy in the human body); (b) drugs; (c) inducing an anti-tumor immune response, or inducing its increase; (d) reducing the number of one or more tumor markers in a patient; (e) stopping or delaying the growth of a tumor or hematologic cancer; (f) discontinuing or delaying the progression of PD-1 related or anti-TIGIT related diseases; (g) stopping or delaying cancer progression; (h) stabilization of PD-1 related diseases or anti-TIGIT diseases; (i) inhibit the growth or survival of tumor cells; (j) removing one or more cancerous lesions or tumors, or reducing their size; (k) reduced progression, onset, or severity of PD-1 related disease or anti-TIGIT disease; (l) reducing the severity or duration of clinical symptoms of PD-1 related or anti-TIGIT related diseases, such as cancer, (m) prolonging the patient's survival compared to expected survival in similar untreated patients, n) cancerous Induction of complete or partial remission of a condition or other PD-1 related disease or anti-TIGIT related disease, o) cancer treatment; Or p) one or more biological agents described herein for (i) use in the treatment of chronic infection, (ii) use as a drug or composition therefor, or (iii) in the manufacture of a drug therefor.

General way

Standard methods in molecular biology are described in [Sambrook, Fritsch and Maniatis (1982 & 1989 2 nd Edition, 2001 3 rd Edition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY]; Sambrook and Russell (2001) Molecular Cloning, 3 ^rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA , Vol. 217, Academic Press, San Diego, Calif.). Standard methods also describe cloning and DNA mutagenesis (Vol 1) in bacterial cells, cloning in mammalian cells and yeast (Vol 2), glycoconjugate and protein expression (Vol 3), and bioinformatics (Vol 4). See Ausbel, et al . (2001) Current Protocols in Molecular Biology, Vols . 1-4 , John Wiley and Sons, Inc. New York, NY.

Methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, are described (Coligan, et al . (2000) Current Protocols in Protein Science, Vol. 1 , John Wiley and Sons, Inc., New York). Chemical analysis, chemical modifications, post-translational modifications, fusion protein preparation, glycosylation of proteins have been described (see, eg, Colingan, et al . (2000) Current Protocols in Protein Science, Vol. 2 , John Wiley and Sons, Inc., New York; Ausubel, et al . (2001) Current Protocols in Molecular Biology, Vol. 3 , John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research , St. Louis, MO; pp. 45-89; see Amersham Pharmacia Biotech (2001) BioDirectory , Piscataway, NJ, pp. 384-391). The preparation, purification, and fragmentation of polyclonal and monoclonal antibodies are described (Coligan, et al . (2001) Current Protocols in Immunology, Vol. 1 , John Wiley and Sons, Inc., New York); Harlow and Lane (1999) Using Antibodies , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra. Standard techniques for characterizing ligand / receptor interactions are available (see, e.g., Colingan, et al . (2001) Current Protocols in Immunology, Vol. 4 , John Wiley, Inc., New York).

Monoclonal, polyclonal, and humanized antibodies can be prepared (see, eg, Sheperd and Dean (eds.) (2000) Monoclonal Antibodies , Oxford Univ. Press, New York, NY); Kontermann and Dubel (eds .) (2001) Antibody Engineering , Springer-Verlag, New York; Harlow and Lane (1988) Antibodies A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 139-243; [Carpenter, et al. . (2000) J. Immunol 165: ... 6205; He, et al (1998) J. Immunol 160: 1029]; [Tang et al (1999) J. Biol Chem 274:... 27371-27378]; Baca et al. (1997) J. Biol . Chem . 272: 10678-10684; Chothia et al. (1989) Nature 342: 877-883; Foote and Winter (1992) J. Mol . Biol . 224: 487-499; see US Pat. No. 6,329,511.

An alternative to humanization is to use a human antibody library displayed on phage or a human antibody library of transgenic mice (Vaughan et al. (1996) Nature Biotechnol. 14: 309-314; Barbas (1995) Nature Medicine 1: 837-839; Mendez et al. (1997) Nature Genetics 15: 146-156; Hoogenboom and Chames (2000) Immunol . Today 21: 371-377; Barbas et al. (2001) Phage Display: A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York]; Kay et al. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual , Academic Press, San Diego, CA]; de Bruin et al. (1999) Nature Biotechnol . 17: 397-399].

Purification of the antigen is not essential for the production of antibodies. Animals can be immunized with cells bearing the antigen of interest. The splenocytes can then be isolated from the immunized animal and the splenocytes can be fused with myeloma cell lines to produce hybridomas (see, eg, Meyaard et al. (1997) Immunity 7: 283-290); Bright et al. (2000) Immunity 13: 233-242; Preston et al. , Supra; Kaithamana et al. (1999) J. Immunol . 163: 5157-5164).

Antibodies can be conjugated, for example, to small drug molecules, enzymes, liposomes, polystyrene glycol (PEG). Antibodies are useful for treatment, diagnostics, kits or other purposes and include, for example, antibodies coupled to dyes, radioisotopes, enzymes, or metals such as colloidal gold (eg, Le Doussal et al. (1991) J. Immunol . 146: 169-175; Gibellini et al. (1998) J. Immunol . 160: 3891-3898; Hsing and Bishop (1999) J. Immunol . 162: 2804-2811. (Everts et al. (2002) J. Immunol . 168: 883-889).

Methods for flow cytometry, including fluorescence activated cell sorting (FACS), are available (see, eg, Owens, et al . (1994) Flow Cytometry Principles for Clinical Laboratory Practice , John Wiley and Sons, Hoboken, NJ); Givan (2001) Flow Cytometry , 2 ^nd ed .; See Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry , John Wiley and Sons, Hoboken, NJ). Fluorescent reagents suitable for modifying nucleic acids, polypeptides, and antibodies, including nucleic acid primers and probes, are available, for example, for use as diagnostic reagents (Molecular Probesy (2003) Catalog , Molecular Probes, Inc., Eugene, OR]; Sigma-Aldrich (2003) Catalogue , St. Louis, MO].

Standard methods of immune system histology have been described (see, eg, Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology , Springer Verlag, New York, NY); Hiatt, et al . (2000) Color Atlas of Histology , Lippincott, Williams, and Wilkins, Phila, PA; see Louis, et al . (2002) Basic Histology: Text and Atlas , McGraw-Hill, New York, NY).

For example, software packages and databases for determining antigen fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments are available (eg, GenBank, Vector NTI® Suite (Vector NTI®). Suite) (Informmax, Inc., Bethesda, Md.); GCG Wisconsin Package (Accelrys, Inc., San Diego, Calif.); DeCypher ) (TimeLogic Corp .: Crystal Bay, Nevada, USA; Menne, et al. (2000) Bioinformatics 16: 741-742); Menne, et al. (2000) Bioinformatics Applications Note 16: 741-742; Wren, et al. (2002) Comput . Methods Programs Biomed . 68: 177-181; von Heijne (1983) Eur. J. Biochem . 133: 17-21; von Heijne ( 1986) Nucleic Acids Res. 14: 4683-4690.

Analytical Method

Suitable analytical methods to assess product stability include size exclusion chromatography (SEC), dynamic light scattering test (DLS), differential scanning calorimetry (DSC), iso-asp quantification, efficacy, UV at 340 nm, UV spectroscopy and FTIR. SEC (J. Pharm. Scien., 83: 1645-1650, (1994)]; Pharm. Res., 11: 485 (1994); J. Pharm. Bio.Anal., 15: 1928 (1997 J. Pharm. Bio.Anal., 14: 1133-1140 (1986)], measures the percentage of monomer in the product and provides information on the amount of soluble aggregates. DSC (Pharm. Res., 15: 200 (1998); Pharm. Res., 9: 109 (1982)) provides information on protein denaturation temperatures and glass transition temperatures. American Lab., November (1991) measures the average diffusion coefficient and provides information on the amount of soluble and insoluble aggregates. UV at 340 nm measures the scattered light intensity at 340 nm and provides information on the amount of soluble and insoluble aggregates. UV spectroscopy measures the absorbance at 278 nm and provides information on protein concentration. FTIR (Eur. J. Pharm. Biopharm., 45: 231 (1998); Pharm. Res., 12: 1250 (1995); J. Pharm. Scien., 85: 1290 (1996)); (J. Pharm. Scien., 87: 1069 (1998)) measure the IR spectrum in the amide 1 region and provide information on protein secondary structure.

Isoquant Isoaspartate Detection System (Promega) is used to measure the iso-asp content in a sample. The kit specifically uses the protein isoaspartyl methyltransferase (PIMT) enzyme to detect the presence of isoaspartic acid residues in the target protein. PIMT catalyzes the transfer of methyl groups from S-adenosyl-L-methionine to isoaspartic acid at the alpha-carboxyl position, resulting in S-adenosyl-L-homocysteine (SAH) in the process. It is relatively small molecule and can generally be isolated and quantified by reverse phase HPLC using the SAH HPLC standard provided in the kit.

The efficacy or bioidentity of an antibody can be measured by its ability to bind an antibody to its antigen. Specific binding of an antibody to its antigen can be quantified by any method known to those skilled in the art, such as immunoassays such as ELISA (enzyme-linked immunosorbent assay).

All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing methods and materials that can be used in connection with the present invention.

While different embodiments of the invention are described herein with reference to the accompanying drawings, the invention is not limited to the precise embodiments described above, and without departing from the scope or spirit of the invention as defined in the appended claims therein. It should be understood that various changes and modifications can be made by those skilled in the art.

Example

Example 1

term- TIGIT  Formulation Buffer  Screening

(1) biophysically / biochemically susceptible; In order to assess (2) pre-formulation (pH, salt and buffer) conditions and (3) compatibility with the entire platform formulation, each anti-TIGIT antibody was selected from the following CDRs: HCDR1 of SEQ ID NO: 108, HCDR2 of SEQ ID NO: 154. A high throughput formulation development study was performed for three anti-TIGIT antibodies with HCDR3 of SEQ ID NO: 110, LCDR1 of SEQ ID NO: 111, LCDR2 of SEQ ID NO: 112, and LCDR3 of SEQ ID NO: 113. Stresses are charged to the molecular surface by size exclusion chromatography (UP-SEC) to detect the formation of high molecular weight species by UV / Vis spectrophotometry on turbidity (A350) as a surrogate for larger aggregates. Determination of sodium dodecyl sulphate capillary electrophoresis (CE-SDS), invisible aggregates to detect proteolytic cleavage of heavy or light chains, by capillary isoelectric focusing (cIEF) to determine the effect on distribution The samples were analyzed by particle analysis, which was not visible to the naked eye.

High throughput formulation screens were formulated with a selection of three buffer species: acetate buffers with pH values ranging from 5.0 to 6.2, citrate buffers with pH values ranging from 5.6 to 6.8, and L-histidine buffers with pH values ranging from 5.0 to 6.8. It consisted of 1 mg / mL anti-TIGIT antibody. Therefore, pH values ranging from 5.0 to 6.8 and ionic strength of 0-150 mM NaCl were investigated. Stress the sample at 50 ° C. for 10 days, and use thermal stability, differential scanning fluorescence (DSF), colloidal stability, Guava (invisible to the naked eye) using size exclusion chromatography (UP-SEC). Aggregation propensity, turbidity (A350 measurement), charge variant profile (cIEF), and calipers were analyzed for fragmentation profiles using flow cytometry based on the relevant characterization assay.

Based on the results obtained from the study, the formulation that gave the protein maximum stability was 10 mM L-histidine with a pH range of 5.6 to 6.2. 10 mM L-histidine with a pH range of 5.6 to 6.2 was monitored by UP-SEC, as compared to SEC Main in the range of -2.0 to -5.0% under different buffer and pH conditions. Minimal aggregation was seen, showing ΔSEC mains in the range -1.85% (data not shown). The cIEF profile showed a decrease in the relative peak areas of the main and basic species, with an increase in the relative peak area of the acidic variants observed for the samples after 10 days at 50 ° C. (cIEF mains -9.0 to -11.3% range). (Data not shown). Reduction of basic variants and increase of acidic variants upon exposure to elevated temperatures are those commonly occurring for mAbs. Salt addition reduced the stability of the protein throughout the studied composition.

Example 2

term- TIGIT  Formulation pH Range Study

In this study, the following CDRs: HCDR1 of SEQ ID NO: 108, HCDR2 of SEQ ID NO: 154, HCDR3 of SEQ ID NO: 110, LCDR1 of SEQ ID NO: 111, LCDR2 of SEQ ID NO: 112, and LCDR3 of SEQ ID NO: 113 Anti-TIGIT antibodies were tested in 10 mM L-histidine buffer at a concentration of 50 mg / mL. 7% (w / v) sucrose (as stabilizer and nonionic tonic modifier) was added to the formulation to increase the bulk stability of the molecule. Anti-TIGIT antibodies were formulated with 10 mM L-histidine buffer, pH 5.5, pH 6.0 and pH 6.5 in 7% sucrose. The stability of the formulation was evaluated as follows:

(1) The stability of the molecules was monitored under accelerated heat and storage stability conditions (up to 6 months at 5 ° C, 25 ° C and 40 ° C) in shading conditions.

(2) Stability studies for freeze-thaw stress and agitation stress were also performed.

(3) Agitation studies were performed on formulations containing various concentrations of polysorbate 80 (PS-80) to assess the concentration of PS-80 in the formulation.

(4) A light stress study was conducted to evaluate the requirements of L-methionine in the formulation.

Substances and Methods

Thermal stability study (3 months)

50 mg / mL anti-TIGIT antibody was formulated at pH 5.5, pH 6.0 or pH 6.5 in 10 mM L-histidine buffer, 7% sucrose, 0.2 mg / mL polysorbate 80. The resulting formulation was sterile filtered, filled into 2R vials, covered with a chlorobutyl stopper stopper and capped with an aluminum cap with seals. Stability studies were performed at 5 ° C. (ambient humidity), 25 ° C. (60% relative humidity) and 40 ° C./(75% relative humidity). UP-SEC, HP-IEX, cIEF (selected samples), MFI, CE-SDS (non-reduced "NR" and reduced "R"), reduced peptide mapping (MFI and reduced peptide mapping are performed at selected time points) Samples were analyzed.

A one month thermal stability study was set up for 25 mg / ml anti-TIGIT antibody formulated with 10 mM L-histidine buffer, 7% sucrose, pH 6.0 in 0.2 mg / ml PS-80. The resulting formulation was sterile filtered, filled into 2R vials, covered with a chlorobutyl stopper stopper and capped with aluminum seals. Stability studies were performed for 1 month at 5 ° C. (ambient humidity), 25 ° C. (60% relative humidity) and 40 ° C. / (75% relative humidity). Samples were analyzed using UP-SEC, cIEF, and CE-SDS (NR and R).

Stirring  Stability studies

50 mg / mL anti-TIGIT antibody was formulated with 10 mM L-histidine buffer, 7% sucrose, pH 6.0 at various concentrations of polysorbate 80 (0, 0.1, and 0.2 mg / mL). The resulting formulation was sterile filtered, filled into 2R vials (1.2 mL fill volume), covered with a chlorobutyl stopper stopper and capped with an aluminum cap with seal. Samples were stirred at 300 RPM for 18 days at 18-22 ° C. in a horizontal position. Samples were analyzed using UP-SEC, MFI, CE-SDS (NR and R).

Freeze-thaw stability

50 mg / mL anti-TIGIT antibody was formulated in 10 mM L-histidine buffer, 7% sucrose, 0.2 mg / mL polysorbate 80, pH 6.0. The resulting formulation was sterile filtered, filled into 2R vials, covered with a chlorobutyl stopper stopper and capped with an aluminum cap with seals. Five freeze-thaw cycles were performed on the samples at −80 ° C. to 18-22 ° C. (at least 24 hours in freezing conditions and at room temperature until complete thawing). Samples were analyzed using UP-SEC, MFI, CE-SDS (NR and R).

Optical stress stability study

Early development studies have shown that some methionine is present, as well as exposed tryptophan residues as readily oxidized under light stress. Formulations containing and without L-methionine under ICH light stress conditions visible light (CWF, 0.1x ICH, 0.2x ICH, 0.5x ICH, 1x ICH) (Formulation 1: 10 mM L-histidine buffer, 7% sucrose, Setup study at 0.2 mg / mL polysorbate 80, pH 6.0 and Formulation 2: 10 mM L-histidine buffer, 10 mM L-methionine, 7% sucrose, 0.2 mg / mL polysorbate 80, pH 6.0) It was. Samples were analyzed using UP-SEC, cIEF, CE-SDS (NR and R) and reduced peptide mapping.

result

Thermal stability results

50 mg / ml Formulation: No significant change was observed after 3 months at all pH values at 5 ° C. in the assay showing stability tested (data not shown). At 25 ° C. and 40 ° C., pH 5.5 and pH 6.0 showed similar stability and the conditions were more stable than pH 6.5 (data not shown). According to UP-SEC, CE-SDS (NR) and cIEF assays at 25 ° C. and 40 ° C., the degradation rate at pH 6.5 was relatively higher than that observed at pH 5.5 and pH 6.0, and also compared with benchmark molecules. Even when relatively high. Therefore, a pH range of 5.5 to 6.0 (pI 8.7) was considered suitable. No oxidation, deamidation or isomerization was observed for anti-TIGIT antibodies after 3 months at all temperature and pH values.

25 mg / ml formulation: Degradation was observed at 25 ° C. and 40 ° C. for all assays tested. The degradation rate was found to be similar to the 50 mg / ml condition (see above).

Stirring  Stability studies

Particles that were visually observed at the end of 7 days were observed in formulations that did not contain Polysorbate 80. Invisible particle analysis showed a significant reduction in particles of 10 μm or greater in formulations containing polysorbate 80 at a concentration of 0.2 mg / mL. No significant difference between the samples was observed when other assays were used.

Freeze-thaw stability

No changes in the stability of the molecules were observed after five freeze-thaw cycles in all assays tested.

Optical stress stability study

When tested by UP-SEC, cIEF, CE-SDS (NR and R), degradation was observed when both formulation samples were exposed to light stress above 0.5 × ICH. Both formulations showed no significant degradation under conditions set up below 0.5 × ICH. Reduced peptide mapping data under light stress conditions of at least 0.5 × showed oxidation of tryptophan and methionine residues. 10 mM L-methionine in the formulation reduced the oxidation level of methionine residues but had no effect on tryptophan oxidation levels.

conclusion

According to the results, 10 mM L-histidine buffer, 10 mM L-methionine, 7% sucrose, 0.2 mg / mL polysorbate 80 pH 5.5-6.0 provides stability and is suitable for supporting shelf life under refrigerated conditions. Is considered.

Example 3

Additional pH Study

Among the six 10 mM histidine buffers of different pH (range of 5.0 to 6.5) the following CDRs: HCDR1 of SEQ ID NO: 108, HCDR2 of SEQ ID NO: 154, HCDR3 of SEQ ID NO: 110, LCDR1 of SEQ ID NO: 111, SEQ ID NO: Anti-TIGIT antibodies were formulated with an LCDR2 of No. 112 and an LCDR3 of SEQ ID NO: 113 on the IgG1 backbone. Thermal stability in different formulations was studied over 8 weeks at 2-8 ° C, 25 ° C and 40 ° C.

Histidine buffers of different pH (5.0-6.5) were prepared by titrating 10 mM L-histidine buffer with 10 mM L-histidine-HCl buffer. Six different histidine buffers with different pH through 4-5 rounds of ultrafiltration using centrifugal apparatus under conditions of 4 ° C. and 4500 rpm-5000 rpm (105-260 min in each round) for anti-TIGIT antibodies Buffer exchange was performed. After buffer exchange, a specific amount of sucrose and polysorbate 80 stock solution (1%, w / w) is added to a solution of different pH to reach the target amount, and an appropriate amount of the corresponding histidine buffer is also added to the antibody The concentration was adjusted to about 50 mg / ml.

The formulation was then filtered in a sterile manner using a 0.22 μm membrane filter. 3 mL of each sample was filled into 6 mL glass vials aseptically for T0, 4 (4W) and 8 (8W) thermal stability studies. 1 mL of each sample was filled into 6 mL glass vials aseptically for a two week (2W) thermal stability study. Filled vials were capped immediately after filling, crimped and sealed. All of the above steps were performed in a biosafety hood. The vials were placed in a cover box and stored at different temperature conditions for thermal stability studies.

Results and discussion

The appearance of all samples remained the same within 4 weeks under all conditions.

However, after 8 weeks, the samples at 2-8 ° C. and 25 ° C. showed a slightly yellowish color, and the samples at 40 ° C. showed a darker yellowish color. During the study period all samples were slightly milky and no particles were observed visually. No significant change in protein concentration of all samples was observed during the study.

Percentage of monomers (%), high molecular weight species (HMW), and percent of late eluting peak (LMW species) were also measured to assess colloidal stability of the sample for purity by size exclusion chromatography (SEC). . Analysis was performed using an Agilent 1260 Infinity system equipped with a TSKgel G3000SWXL size exclusion chromatography column (300 7.8 mm, 5 μm). Mobile phase was 50 mM PB, 300 mM NaCl, pH 7.0 ± 0.2 and flow rate was set to 1.0 mL / min. Samples were diluted to 10 mg / mL for injection and detected at 280 nm using a UV detector.

UPSEC data is presented in the table below:

As shown in the table above, the SEC main peak (%) was stable at 2-8 ° C. in all samples, but a significant decrease in main peak (%) was observed at 25 ° C. and 40 ° C. The rate of decrease of the main peak (%) in the sample was faster at 40 ° C. than at 25 ° C. For 8 weeks at 40 ° C., HMW% was greater in pH 6.2 and 6.5 samples, while LMW% was greater in pH 5.0 and 5.3 samples.

To assess the chemical stability of the formulation, capillary isoelectric focusing (cIEF) was performed to assess chemical stability and to monitor the change in charge variant profile over time. Briefly, a 20 μl (2.0 mg / mL) reference standard or sample was prepared with 0.5 μl pi 5.85 marker, 0.5 μl pi 9.77 marker, 1 μl Pharmalyte 3-10, 0.5 μl Pharmalite 5- 8, 0.5 μl of Pharmalite 8-10.5, 35 μl of 1% methyl cellulose, 37.5 μl of 8 M urea. Purified water was added until the final volume was 100 μl. The mixture was then analyzed using an iCE-3 capillary isoelectric focusing analyzer equipped with a fluorocarbon coated full column detection capillary. Step 2: Focusing was performed for (1) 1 min at 1.5 kV and (2) 8 min at 3 kV. During the experiment, the automatic sampler tray was kept at 5 ° C.

CIEF data assessing acidic variant, major peak (%), and basic variant (%) levels are in the table below.

As can be seen from the table above, the cIEF main peak (%), acidic peak (%) and basic peak (%) at 2-8 ° C. were cross-linked stable and similar in all samples.

At 2-8 ° C., cIEF main basic peak (%) also increased; The increase in pH 5.0 and 5.3 buffer samples was greater than the increase in other samples. At 25 ° C., the major peak (%), acidic peak (%) and basic peak (%) were stable for a period within the first two weeks, but changed slightly after four weeks, where the major peak (%) decreased While the acidic peak (%) increased accordingly. In different formulations this rate of change was similar. At 40 ° C., even after two weeks, a significant decrease in major peak (%) and a significant increase in acidic peak (%) were observed in all formulations, but the extent of change was similar in each formulation. Basic peak (%) also increased; The increase in pH 5.0 and 5.3 buffer samples was greater than the increase in other samples.

To assess the purity of the formulation, a non-reduced caliper assay was performed. Briefly, the purchased sample buffer was mixed with a 10% sodium dodecyl sulfate (SDS) solution at a volume ratio of 20 to 1, and a 100 mM N-ethylmaleimide solution was added to the mixed solution at a volume ratio of 0.7 to 20 ( Sample denatured solution). The standard or sample was first diluted to 1 mg / mL and 2 μl of diluted standard or sample was mixed with 7 μl of sample denaturing solution. The mixture was incubated at 70 ° C. for 10 min. 35 μl of purified water was added to the incubated solution and 42 μl of mixed solution was transferred to a 96 well plate for analysis. Sample plates were analyzed with LabChip GX II HT using the HT Antibody Analysis 200 assay.

Non-reduced caliper assay data evaluating purity (%) are presented in the table below:

As shown in the table above, for 8 weeks at -2-8 ° C, the caliper_non-reduced purity of each formulation was relatively stable. For 8 weeks at 25 ° C., the purity of each formulation slightly decreased. At 40 ° C., the purity decreased significantly, especially when the sample was in pH 5.0 and 5.3 buffer, the decrease appeared much faster than the others. Molecular size was stable during the study (data not shown).

To further assess the purity of the formulation, a reduced caliper assay was also performed. Briefly, the purchased sample buffer was mixed with a 10% SDS solution at a volume ratio of 20 to 1 and 1 M dithiothreitol solution was added to the mixed solution at a volume ratio of 0.7 to 20 (referred to as sample denaturation solution). The standard or sample was first diluted to 1 mg / mL and 2 μl of diluted standard or sample was mixed with 7 μl of sample denaturing solution. The mixture was incubated at 70 ° C. for 10 min. 35 μl of purified water was added to the incubated solution and 42 μl of mixed solution was transferred to a 96 well plate for analysis. Sample plates were analyzed with Labchip GX II HT using the HT Antibody Analysis 200 assay.

Reduced caliper analysis data to evaluate the purity (%) is presented in the table below:

As can be seen above, for 8 weeks at 2-8 ° C and 25 ° C, the caliper_reduced purity of each formulation was relatively stable. At 40 ° C., a marked decrease in caliper_R purity was observed in all formulations. In pH 5.0 buffer, the greatest decrease in purity of the sample was followed by the sample in pH 5.3 buffer. The reduction rates in pH 5.6, 5.9 and 6.2 buffers were similar but appeared slower. The purity of the sample in pH 6.5 buffer showed the slowest decrease. Purity reduction was possibly due to the light chain (LC)% being stable while the heavy chain (HC)% decreased. Antibody light and heavy chain sizes were stable in all samples over 8W.

Example 4

Methionine Free  term- TIGIT  Formulation

The following CDRs: anti-TIGIT having HCDR1 of SEQ ID NO: 108, HCDR2 of SEQ ID NO: 154, HCDR3 of SEQ ID NO: 110, LCDR1 of SEQ ID NO: 111, LCDR2 of SEQ ID NO: 112, and LCDR3 of SEQ ID NO: 113 Antibodies were tested in 10 mM L-histidine buffer, 7% sucrose, 0.2 mg / mL PS-80 at a pH ranging from 5.0 to 6.5 at a concentration of 50 mg / mL. The stability of the molecules was monitored under accelerated heat and storage stability conditions in shading conditions. In addition to thermal stability, freeze-thaw stability, stirring stability, and optical stress stability studies were also conducted. Stability was tested including UP-SEC, cIEF, CE-SDS, MFI and reduced peptide mapping.

result

Thermal Stability Study (8 weeks)

In all assays showing stability tested, the anti-TIGIT antibodies were stable in all susceptible properties tested at 5 ° C. Degradation rates were higher at 40 ° C. than at 25 ° C., as observed using UP-SEC, Caliper CE-SDS, cIEF, MFI. At 25 ° C. and 40 ° C. the following results were noteworthy:

UP-SEC: A monomer (%) decrease was observed for all pH values from 5.0 to 6.5. The major decrease in peak at lower pH values (5.0 and 5.3) is mainly due to the increase in low molecular weight (LMW) species (%), whereas the decrease in monomer (%) at pH 6.5 is primarily high molecular weight (HMW) species (%) It was because of the increase. Monomer (%) reduction was highest at pH 6.5 after 8 weeks of accelerated stability (data not shown)

cIEF: cIEF main peak reduction was observed for all pH values of 5.0 to 6.5 at 25 ° C and 40 ° C. The major peak reduction at higher pH values (6.3-6.5) was mainly due to the increase in acidic variants, while the major peak reduction at pH 5.0 and 5.3 was due to the increase in both basic variants as well as basic variants (data is Not shown).

CE-SDS (Caliper): Non-reduced CE-SDS major peak reduction was observed mainly at 40 ° C. due to the presence of fragmented species. Formulations with lower pH values (5.0 and 5.3) showed relatively higher fragmentation rates than those of the remaining pH values.

The reduced CE-SDS (caliper) analysis showed that for 8 weeks at 5 ° C and 25 ° C, the purity of each formulation was relatively stable. At 40 ° C., a marked decrease in purity was observed in all formulations. In pH 5.0 buffer, the greatest decrease in purity of the sample was followed by the sample in pH 5.3 buffer.

MFI: Invisible particle growth was observed in all formulations at 40 ° C. The increase in invisible particles occurred highest at pH 6.3 and 6.5 compared to the rest of the buffer.

Reduced Peptide Mapping: Among the vulnerable properties identified, only M254 showed a relative increase in oxidation after 8 weeks at 40 ° C. when compared to the initial sample.

Conclusions: 10 mM L-histidine buffer, 7% sucrose, 0.2 mg / mL PS-80, pH 5.6-6.3 adequately supported the storage stability of anti-TIGIT antibodies for 8 weeks.

Stirring Stability Study

100 RPM at 18-22 ° C. with 50 mg / mL anti-TIGIT formulation (in any of 10 mM L-histidine buffer, 7% sucrose, pH 5.8 and 0, 0.1, 0.2 and 0.3 mg / mL PS-80) When lightly stirred for up to 7 days, no change was observed in soluble aggregates, charged variants, fragmentation or particles which were not visible to the naked eye.

Freeze-thaw stability

When five freeze / thaw cycles were performed (-80 ° C.) on 50 mg / mL anti-TIGIT antibody (in 10 mM L-histidine buffer, 7% sucrose, 0.2 mg / mL PS-80, pH 5.8) Freezing for 2 hours and thawing for 1 hour at room temperature), no changes were observed in soluble aggregates, charged variants, fragmentation or invisible particles.

Optical stress stability study

Visible light stress (5000 lx) was applied to the 50 mg / ml formulation for 48 hours. Under these conditions, soluble aggregates, charged variants, invisible particles, pH, concentration, fragmentation, and oxidation changed to a minimum under ˜0.2 × ICH conditions (12H light exposure). Under ˜1 × (48H light exposure) conditions soluble aggregates, acidic variants, fragmentation, invisible particles and methionine oxidation increased.

conclusion

Based on this study, 10 mM L-histidine buffer, 7% sucrose, 0.2 mg / mL PS-80 pH 5.3-6.3 could support the stability of anti-TIGIT antibodies. Methionine oxidation was observed upon exposure to extreme light stress. As mentioned in Example 2, addition of 10 mM L-methionine reduces the oxidation of methionine residues.

Example 5

Polysorbate  80 screening

The following CDRs: anti-TIGIT having HCDR1 of SEQ ID NO: 108, HCDR2 of SEQ ID NO: 154, HCDR3 of SEQ ID NO: 110, LCDR1 of SEQ ID NO: 111, LCDR2 of SEQ ID NO: 112, and LCDR3 of SEQ ID NO: 113 Antibodies were formulated into four (4) formulations of pH 5.8, 10 mM L-histidine buffer with different concentrations of PS-80 (as shown below). Protein stability in different formulations was studied in agitation or non-cut conditions over a 7 day period at 20 ° C.

Formulations were formulated to pH 5.8 in 10 mM L-histidine buffer using a laboratory scale TFF buffer exchange system. The formulated protein, differing in polysorbate 80 content, was then filtered in a sterile manner using a 0.22 μm membrane filter. 2 mL of each sample was then filled into 6 mL glass vials in a sterile manner. Filled vials were capped immediately after filling, crimped and sealed. The sample was divided into a stirring group and a control group. In the stirring group, the vial was transferred to a cover box and then placed in a thermostat shaker and stirred at 20 ° C. at 100 rpm for up to 7 days. In the control group, the vial was transferred to a cover box and placed in a thermostat shaker, provided the shaker remained at 20 ° C. for up to 7 days without moving.

After 3 and 7 days, the antibody stability in the different formulations that were stirred or compared was studied.

UPSEC data assessing high molecular weight species (HMW or aggregates), monomer (%), and LMW (low molecular weight species) levels are in the table below:

As can be seen, the polysorbate 80 content did not have a significant effect on SEC purity under agitation or comparison conditions. The polysorbate 80 content did not have a significant effect on the ratio of the main peak, acidic peak, and basic peak in the pI, cIEF assay under agitation or comparative conditions for up to 7 days.

As shown in the table below, the polysorbate 80 content did not significantly affect caliper_non-reduced purity in agitation or control panel for up to 7 days.

Reduced caliper analysis was also performed. The PS-80 content did not have a significant effect on caliper_reduced purity for 7 days under agitation or counterpart conditions.

In order to measure particles that were not visible to the naked eye, about 1500 μl of each sample was taken from the glass vial container and tested by microflow imaging (MFI) according to the user's manual. Particle concentrations of different size ranges were recorded, including 1-2 μm, 2-5 μm, 5-10 μm, 10-25 μm and> 25 μm (see below). The polysorbate 80 content did not have a significant effect on the particle concentration under agitation or control panel conditions for up to 7 days.

Example 6

Chelator  adding

In this study, 10 mM L-histidine buffer (pH = 5.8), 0.02% (w / v) polysorbate 80, 10 mM L-methionine ("L-Met") in the presence or absence of 20 uM or 50 uM DTPA. , The following CDRs in 7% w / v sucrose: HCDR1 of SEQ ID NO: 108, HCDR2 of SEQ ID NO: 154, HCDR3 of SEQ ID NO: 110, LCDR1 of SEQ ID NO: 111, LCDR2 of SEQ ID NO: 112, and SEQ ID NO: The stability of anti-TIGIT antibodies with LCDR3 of number 113 was compared.

Three formulations were filled into vials and performed for stability for 18 weeks at 5 ° C. (ambient humidity), 25 ° C. (60% relative humidity), and 40 ° C. (75% relative humidity) in a shaded state.

The percentage of monomers as well as the percentage of high molecular weight species (HMW) and late eluting peaks (LMW species) were measured to assess colloidal stability of the sample for purity by size exclusion chromatography (SEC). It was. UPSEC data to evaluate% HMW (aggregate), monomer (%), and% LMW levels are in the table below:

As shown in the table above, the propensity to increase the% HMW peak and% LMW peak for all three formulations at 5 ° C, 25 ° C, and 40 ° C for up to 18 weeks (and consequently, decrease in monomer peak (%)). Appeared to be. Compared to 40 ° C., both formulations showed smaller trends at 25 ° C., but showed similar trends. No substantial change was observed at 5 ° C. Compared to Formulation 2 (20 uM DTPA) and Formulation 3 (50 uM DTPA), Formulation 1 shows a greater increase in% HMW and% LMW. In addition, Formulation 1 showed a greater reduction in monomer (%) when compared to Formulations 2 and 3. Similar results were observed in HP-IEX analysis (data not shown).

To assess whether DTPA can protect the formulation from oxidative stress, three formulations were filled into vials and exposed to light (0.5 × ICH and 1 × ICH). As can be seen in the table below, Formulation 1 is in oxidation (%) of M254, M430 and W104 (methionine and tryptophan susceptible to oxidation) compared to Formulation 2 (20 uM DTPA) and Formulation 3 (uM DTPA). The increase is even greater. Thus, DTPA can further improve the stability of anti-TIGIT antibody formulations.

Example 7

term- TIGIT  Long-term Stability of Antibody Formulations

This example describes long term stability data for anti-TIGIT antibodies formulated in L-histidine buffer, L-methionine, sucrose, polysorbate 80 and water for injection as follows:

The solution was filled into a USP Type 1 glass vial with elastomeric stopper and aluminum seal. The vials were then incubated at three different storage conditions: 5 ° C. (ambient humidity), 25 ° C. (60% relative humidity), and 40 ° C. (75% relative humidity). For all storage conditions, time points 0, 1 month, 3 months, 6 months, 9 months (5 ° C and 25 ° C storage conditions), 12 months (5 ° C and 25 ° C storage conditions), 18 months (5 ° C) Storage conditions), 24 months (5 ° C. storage conditions) and 36 months (5 ° C. conditions) are collected.

result

This result demonstrates the overall physical and chemical stability of the anti-TIGIT antibody when stored at the recommended long term conditions at 5 ° C. for 18 months. There was no measurable loss of efficacy observed under the recommended storage conditions and purity was within the specification specification. The results are shown in the table below:

Protein concentration

Protein concentration stability data for all time points and conditions did not show any noticeable changes as a function of storage time or condition, and all results were within the acceptable range of 45-55 mg / ml.

pH

There was no significant change in pH at 5 ° C, 25 ° C, and 40 ° C conditions. 1 shows pH data from time point 0 to 9 months.

Polysorbate  80

The polysorbate 80 content at the recommended storage conditions of 5 ° C. was slightly reduced to 0.13 mg / ml at 9 and 18 months (18 months data not shown). It was observed that polysorbate 80 exhibited a decreasing tendency at 25 ° C. (acceleration) and 40 ° C. (stressed). At 40 ° C., the polysorbate 80 concentration decreased to 0.06 mg / ml at 6 months, and at 25 ° C., the polysorbate 80 concentration decreased to 0.07 mg / ml at 9 months. Polysorbate 80 concentration data for up to 9 months is shown in FIG. 2.

Binding efficacy by ELISA

The observed ELISA binding results showed no clear tendency at any time or condition. Efficacy data for up to 9 months is shown in FIG. 3.

Purity by UP-SEC

Data for purity by UP-SEC for up to 9 months are shown in FIG. 4 for monomer (%), FIG. 5 for high molecular weight species (%), and FIG. 6 for low molecular weight species (%). Is shown.

At the recommended storage conditions of 5 ° C., the monomer (%) is slightly reduced and the corresponding high molecular weight species (%) is slightly increased and stable over 18 months. From early to 18 months, low molecular weight species (%) are below the quantitative limit of 0.4% (<QL). At 25 ° C. conditions, monomer (%) decreased from early to 12 months and correspondingly high molecular weight species (%) increased. At 9 and 12 months, low molecular weight species (%) were reported to be above QL.

Under stressed conditions at 40 ° C., monomer (%) decreased from 98.7% to 93.8%, correspondingly high molecular weight species increased from 1.33% to 2.63% and low molecular weight species increased from <QL to 3.53% It was. These results were not unexpected given the storage conditions.

Reduced and Non-reducing CD-SDS

7 and 8 show purity data for up to 9 months as measured by reduced and non-reduced CD-SDS. There was no noticeable propensity in reduced (heavy and light chain (%)) or unreduced (intact IgG (%)) CE-SDS at long term storage conditions of 5 ° C., and the result was ≥ GMP drug product acceptance criteria. It was in the 90.0% range. At accelerated 25 ° C. conditions, decreasing tendency was observed for non-reduced conditions. Decreasing tendency was also observed for reduced CE-SDS conditions. In both cases of reduced and non-reduced CE-SDS, all results up to 9 months were within the GMP acceptance criteria. At the stressed conditions of 40 ° C., at 6 months, the reduced and non-reduced CD-SDS results were both below ≧ 90.0% acceptance criteria set for the GMP drug product. The non-reduced CE-SDS results were 88.9% at 3 months, out of specification and then decreased to an additional 79.6% at 6 months. For reduced CE-SDS, the heavy and light chains (%) decreased from 94.2% at 3 months to 87.7% at 6 months. This reduction at 40 ° C. was not unexpected given the nature of the conditions.

HP- On IEX  Charge Variant

At 5 ° C. (long term storage), the acidic variant (%) initially increased slightly from 21.46% to 22.49% at 9 months, and correspondingly the total major value decreased slightly from 68.8% to 67.1% at 9 months. Basic variants (%) begin to increase slightly at 9 months, increasing from 10.15% at 6 months to 10.38% at 9 months. At 25 ° C. (acceleration), the total major value decreased from 68.8% at initial time point to 47.6% at 9 months. As the total major value decreased, correspondingly, the acidic variant increased from 21.46% to 39.86% and the basic variant increased slightly from 9.70% to 11.51%. At 40 ° C. (stressed), the total major value decreased significantly to 10.1% at 6 months, correspondingly correspondingly increasing to 80.02% for acidic variants and 9.95% for basic variants.

Particulate matter

Particulate matter was measured by mHIAC. The results at 5 ° C. conditions from the beginning to the ninth month were much lower than the acceptance criterion: ≦ 6,000 particles per container when ≧ 10 μm and ≦ 600 particles per container when ≧ 25 μm. At 25 ° C., particles with ≧ 10 μm were reported to increase from 13 particles per container at the initial time point to 460 particles per container at 9 months. For ≧ 25 μm microparticles, the particles were reduced, resulting in 3 particles per container at 9 months. 25 ° C. data at all time points were within acceptable limits for both ≧ 10 μm and ≧ 25 μm analyzes. Data at 40 ° C. showed a sharp increase in ≧ 10 μm particles with 9,258 particles per container at 9 months. These results deviate from the acceptance criteria of ≤ 600 particles per container. The results for ≧ 25 μm particles increased to 124 particles per container at the 9 month stability point while meeting the ≧ 25 μm acceptance criteria (≦ 600 particles per container).

Turbidity

Turbidity was measured from the spectral absorbance at 350 nm. Under longer term storage conditions, there were no significant changes at storage conditions up to 9 months at 5 ° C. At 25 ° C. conditions, at 3 months the results increased slightly, to 0.163 AU, and by 9 months the results continued to increase to 0.196 AU. At 40 ° C., there was a more significant increase, starting at 1 month at 0.188 AU, followed by a significant increase to 0.453 AU by 9 months.

conclusion

Based on this data, pH, protein concentration, appearance and visual observation of particles (data not shown) and potency and particulate matter (data not shown) at storage conditions of 5 ° C. during the stability study on test day 18 months. No major change or propensity was observed. The exception is that the color is slightly darker, the PS-80 content is reduced to 0.13 mg / ml, and no noticeable change or propensity was observed in any stability test at 5 ° C.

Based on data for long term stability at 5 ° C., the expected shelf life of anti-TIGIT formulations containing L-histidine buffer, sucrose, polysorbate 80 and L-methionine is 30 months. Formulations further comprising chelators are expected to reduce the degradation of the observed polysorbate 80.

Example 8

term- TIGIT  Antibodies and Anti-PD-1 Antibodies Complex formulation

Co-formulation of two antibodies into a single agent is more convenient for the patient and increases compliance with the administration of the two antibodies together. Co-formulation of two antibodies into a single agent is more convenient for the patient and increases compliance with the administration of the two antibodies together. On the IgG1 backbone, the following CDRs: HCDR1 of SEQ ID NO: 108, HCDR2 of SEQ ID NO: 154, HCDR3 of SEQ ID NO: 110, LCDR1 of SEQ ID NO: 111, LCDR2 of SEQ ID NO: 112, and LCDR3 of SEQ ID NO: 113 Eggplants were co-formulated with anti-TIGIT antibodies with pembrolizumab. Based on protein-protein interactions (shown below), the co-formulations (shown below) were shown to be stable over pH 5.0-6.0. Therefore, the combination formulation (P1T1) was selected at pH 5.0, 5.5 and 6.0 and evaluated for further thermal stability of 5 ° C, 25 ° C and 40 ° C with two controls (PD1 antibody and anti-TIGIT antibody).

The formulations were prepared as liquid formulations as follows:

Each formulation was charged to 1 mL in a 2R vial. Stability by visual inspection, protein concentration, microflow imaging (MFI) (particulate evaluation), mixed mode size exclusion chromatography (SEC) (aggregation evaluation), IEX (charge variant evaluation), and UP-SEC (aggregation evaluation) Will measure. The thermal stability protocol is as follows:

Protein-protein interactions showing colloidal and thermal stability of different co-formulations were measured. Repulsive protein-protein interactions, where the diffusion interaction parameter (K _D ) value is positive (K _D > 0), indicate a stable formulation with low propensity to aggregate. It has been shown that the Kd of the co-formulations has a positive K _D value indicating repulsion and stabilizing protein-protein interactions, suggesting that the cohesion tends to be lower and stable co-formulations.

Based on the diffusion interaction parameter (K _D ) value being a positive value or K _D > 0, when co-formulated, the antibody will act and exhibit a good response when co-formulated together, similar to a single antibody formulation. It is expected.

                         SEQUENCE LISTING <110> Narasimhan, Chakravarthy Nachu        De, Arnab   <120> STABLE FORMULATIONS OF ANTI-TIGIT ANTIBODIES ALONE AND IN        COMBINATION WITH PROGRAMMED DEATH RECEPTOR 1 (PD-1) ANTIBODIES        AND METHODS OF USE THEREOF <130> 24453 <150> 62 / 500,278 <151> 2017-05-02 <160> 293 <170> PatentIn version 3.5 <210> 1 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 1 Arg Ala Ser Lys Gly Val Ser Thr Ser Gly Tyr Ser Tyr Leu His 1 5 10 15 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 2 Leu Ala Ser Tyr Leu Glu Ser 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 3 Gln His Ser Arg Asp Leu Pro Leu Thr 1 5 <210> 4 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 4 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser             20 25 30 Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro         35 40 45 Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala     50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg                 85 90 95 Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 110 <210> 5 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 5 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser             20 25 30 Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro         35 40 45 Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala     50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg                 85 90 95 Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg             100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln         115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr     130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr                 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys             180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro         195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys     210 215 <210> 6 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 6 Asn Tyr Tyr Met Tyr 1 5 <210> 7 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 7 Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe Lys 1 5 10 15 Asn      <210> 8 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 8 Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr 1 5 10 <210> 9 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 9 Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr             20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe     50 55 60 Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln             100 105 110 Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 10 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 10 Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr             20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe     50 55 60 Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln             100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val         115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala     130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val                 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro             180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys         195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro     210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr                 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu             260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys         275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser     290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile                 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro             340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu         355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn     370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg                 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu             420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys         435 440 445 <210> 11 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 11 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 12 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 12 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 13 Gln Gln Ser Ser Asn Trp Pro Arg Thr 1 5 <210> 14 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 14 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile         35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 <210> 15 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 15 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile         35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 16 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 16 Asn Ser Gly Met His 1 5 <210> 17 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 17 Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly      <210> 18 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 18 Asn Asp Asp Tyr One <210> 19 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 19 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser             20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser             100 105 110 Ser      <210> 20 <211> 440 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 20 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser             20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser             100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser         115 120 125 Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp     130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr                 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys             180 185 190 Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp         195 200 205 Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala     210 215 220 Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 225 230 235 240 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val                 245 250 255 Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val             260 265 270 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln         275 280 285 Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln     290 295 300 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 305 310 315 320 Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro                 325 330 335 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr             340 345 350 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser         355 360 365 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr     370 375 380 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 385 390 395 400 Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe                 405 410 415 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys             420 425 430 Ser Leu Ser Leu Ser Leu Gly Lys         435 440 <210> 21 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 21 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Phe Ser Tyr Leu His 1 5 10 15 <210> 22 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 22 Leu Ala Ser Asn Leu Glu Ser 1 5 <210> 23 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 23 Gln His Ser Trp Glu Leu Pro Leu Thr 1 5 <210> 24 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 24 Ser Tyr Tyr Leu Tyr 1 5 <210> 25 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 25 Gly Val Asn Pro Ser Asn Gly Gly Thr Asn Phe Ser Glu Lys Phe Lys 1 5 10 15 Ser      <210> 26 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 26 Arg Asp Ser Asn Tyr Asp Gly Gly Phe Asp Tyr 1 5 10 <210> 27 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 27 Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr             20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe     50 55 60 Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln             100 105 110 Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 28 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 28 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser             20 25 30 Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro         35 40 45 Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala     50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg                 85 90 95 Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 110 <210> 29 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 29 Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser             20 25 30 Gly Tyr Ser Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro         35 40 45 Gln Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Asp     50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser 65 70 75 80 Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln His Ser Arg                 85 90 95 Asp Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 110 <210> 30 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 30 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser             20 25 30 Gly Tyr Ser Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro         35 40 45 Gln Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Asp     50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Lys Ile Ser 65 70 75 80 Arg Val Glu Ala Glu Asp Val Gly Leu Tyr Tyr Cys Gln His Ser Arg                 85 90 95 Asp Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 110 <210> 31 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 31 Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr             20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe     50 55 60 Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln             100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val         115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala     130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val                 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro             180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys         195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro     210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr                 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu             260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys         275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser     290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile                 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro             340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu         355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn     370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg                 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu             420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys         435 440 445 <210> 32 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 32 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser             20 25 30 Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro         35 40 45 Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala     50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg                 85 90 95 Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg             100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln         115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr     130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr                 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys             180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro         195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys     210 215 <210> 33 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 33 Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser             20 25 30 Gly Tyr Ser Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro         35 40 45 Gln Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Asp     50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser 65 70 75 80 Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln His Ser Arg                 85 90 95 Asp Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg             100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln         115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr     130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr                 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys             180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro         195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys     210 215 <210> 34 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 34 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser             20 25 30 Gly Tyr Ser Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro         35 40 45 Gln Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Asp     50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Lys Ile Ser 65 70 75 80 Arg Val Glu Ala Glu Asp Val Gly Leu Tyr Tyr Cys Gln His Ser Arg                 85 90 95 Asp Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg             100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln         115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr     130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr                 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys             180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro         195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys     210 215 <210> 35 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 35 Ser Asp Tyr Trp Gly 1 5 <210> 36 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 36 Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 37 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 37 Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly Tyr 1 5 10 15 Phe Asp Phe              <210> 38 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 38 Lys Ala Ser Gln Ser Ile His Lys Asn Leu Ala 1 5 10 <210> 39 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 39 Tyr Ala Asn Ser Leu Gln Thr 1 5 <210> 40 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 40 Gln Gln Tyr Tyr Ser Gly Trp Thr 1 5 <210> 41 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 41 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Ser Ser Ile Ala Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Lys Phe Pro Gly Asn Lys Met Glu Trp Met         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu 65 70 75 80 Gln Leu His Ser Val Thr Thr Asp Asp Thr Ala Thr Tyr Ser Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Pro Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 42 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 42 Asp Ile Gln Met Thr Gln Ser Pro Ser Leu Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Leu Asn Cys Lys Ala Ser Gln Ser Ile His Lys Asn             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Leu Gly Glu Ala Pro Lys Phe Leu Ile         35 40 45 Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr Ser Gly Trp Thr                 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Leu Lys             100 105 <210> 43 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 43 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 44 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 44 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 45 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 45 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 46 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 46 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ser Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 47 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 47 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu His Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 48 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 48 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu His Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 49 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 49 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ser Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu His Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 50 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 50 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ser Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Ile Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu His Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 51 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 51 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 52 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 52 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 53 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 53 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 54 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 54 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Ser Ser Ile Ser Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 55 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 55 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu His Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 56 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 56 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu His Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 57 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 57 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Ser Ser Ile Ser Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu His Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 58 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 58 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Ser Ser Ile Ser Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu His Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 59 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 59 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Trp Thr                 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 60 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 60 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile         35 40 45 Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Trp Thr                 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 61 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 61 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile         35 40 45 Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Trp Thr                 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 62 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 62 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile         35 40 45 Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Trp Thr                 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 63 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 63 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Phe Leu Ile         35 40 45 Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Trp Thr                 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 64 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 64 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Phe Leu Ile         35 40 45 Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Trp Thr                 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 65 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 65 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ala Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala         115 120 125 Ser      <210> 66 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 66 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ala Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 67 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 67 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ala Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Lys Pro Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 68 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 68 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ala Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Lys Leu Glu Trp Ile         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 69 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 69 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ala Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Met Glu Trp Ile         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 70 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 70 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ala Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Lys Pro Pro Gly Lys Lys Met Glu Trp Ile         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 71 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 71 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ala Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Gln Phe Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Asp Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 72 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 72 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ala Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Lys Pro Pro Gly Lys Lys Met Glu Trp Ile         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 73 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 73 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Ser Ser Ile Ala Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 74 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 74 Glu Val Gln Leu Gln Gln Ser Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Ser Ser Ile Ala Ser Asp             20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 75 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 75 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Pro Gly 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Ser Ser Ile Ala Ser Asp             20 25 30 Tyr Trp Gly Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 125 <210> 76 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 76 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Trp Thr                 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 77 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 77 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile         35 40 45 Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Trp Thr                 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 78 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 78 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr Ser Gly Trp Thr                 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 79 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 79 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile         35 40 45 Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr Ser Gly Trp Thr                 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 80 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 80 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Trp Thr                 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 81 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 81 Gly Tyr Ser Ile Thr Ser Asp Tyr Ala Trp Asn 1 5 10 <210> 82 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 82 Tyr Ile Ser Asn Ser Gly Ser Ala Ser Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 83 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 83 Leu Ile Tyr Tyr Asp Tyr Gly Gly Ala Met Asn Phe 1 5 10 <210> 84 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 84 Lys Ala Ser Gln Gly Val Ser Thr Thr Val Ala 1 5 10 <210> 85 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 85 Ser Ala Ser Tyr Arg Tyr Thr 1 5 <210> 86 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 86 Gln His Tyr Tyr Ser Thr Pro Trp Thr 1 5 <210> 87 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 87 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp             20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp         35 40 45 Met Gly Tyr Ile Ser Asn Ser Gly Ser Ala Ser Tyr Asn Pro Ser Leu     50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys                 85 90 95 Ala Thr Leu Ile Tyr Tyr Asp Tyr Gly Gly Ala Met Asn Phe Trp Gly             100 105 110 Gln Gly Thr Ser Val Thr Val Ser Ser         115 120 <210> 88 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 88 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Gly Val Ser Thr Thr             20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile         35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Val Tyr Tyr Cys Gln His Tyr Tyr Ser Thr Pro Trp                 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 89 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 89 Tyr Ala Ser Asn Leu Gln Thr 1 5 <210> 90 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 90 Tyr Ala Ser Ser Leu Gln Thr 1 5 <210> 91 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 91 Tyr Ala Ser Thr Leu Gln Thr 1 5 <210> 92 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 92 Tyr Ala Thr Thr Leu Gln Thr 1 5 <210> 93 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 93 Tyr Ala Ser Tyr Leu Gln Thr 1 5 <210> 94 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 94 Tyr Ala Asn Gln Leu Gln Thr 1 5 <210> 95 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 95 Tyr Ala Gly Ser Leu Gln Thr 1 5 <210> 96 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 96 Tyr Ala Ser Gln Leu Gln Thr 1 5 <210> 97 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 97 Tyr Ala Asp Ser Leu Gln Thr 1 5 <210> 98 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 98 Gln Gln Tyr Tyr Ser Gly Phe Thr 1 5 <210> 99 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 99 Gln Gln Tyr Tyr Ser Gly Tyr Thr 1 5 <210> 100 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 100 Gln Gln Tyr Tyr Ser Gly Ile Thr 1 5 <210> 101 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 101 Gln Gln Tyr Tyr Ser Gly Val Thr 1 5 <210> 102 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 102 Gln Gln Tyr Tyr Ser Gly Leu Thr 1 5 <210> 103 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 103 Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Phe Glu Gly Tyr 1 5 10 15 Phe Asp Phe              <210> 104 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 104 Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Tyr Glu Gly Tyr 1 5 10 15 Phe Asp Phe              <210> 105 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 105 Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Ile Glu Gly Tyr 1 5 10 15 Phe Asp Phe              <210> 106 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 106 Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Val Glu Gly Tyr 1 5 10 15 Phe Asp Phe              <210> 107 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 107 Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Leu Glu Gly Tyr 1 5 10 15 Phe Asp Phe              <210> 108 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 108 Ser Tyr Val Met His 1 5 <210> 109 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 109 Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly      <210> 110 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 110 Gly Gly Pro Tyr Gly Trp Tyr Phe Asp Val 1 5 10 <210> 111 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 111 Arg Ala Ser Glu His Ile Tyr Ser Tyr Leu Ser 1 5 10 <210> 112 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 112 Asn Ala Lys Thr Leu Ala Glu 1 5 <210> 113 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 113 Gln His His Phe Gly Ser Pro Leu Thr 1 5 <210> 114 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 114 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr             20 25 30 Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Gly Pro Tyr Gly Trp Tyr Phe Asp Val Trp Gly Ala Gly             100 105 110 Thr Thr Val Thr Val Ser Ser         115 <210> 115 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 115 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu His Ile Tyr Ser Tyr             20 25 30 Leu Ser Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val         35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His His Phe Gly Ser Pro Leu                 85 90 95 Thr Phe Gly Ala Gly Thr Thr Leu Glu Leu Lys             100 105 <210> 116 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 116 Tyr Ile Asp Pro Tyr Asn Arg Gly Ala Lys Tyr Asn Glu Lys Phe Gly 1 5 10 15 <210> 117 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 117 Tyr Ile Asp Pro Tyr Asn Leu Gly Ala Lys Tyr Asn Glu Lys Phe Gly 1 5 10 15 <210> 118 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 118 Tyr Ile Asp Pro Tyr Asn Lys Gly Ala Lys Tyr Asn Glu Lys Phe Gly 1 5 10 15 <210> 119 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 119 Tyr Ile Asp Pro Tyr Asn Phe Gly Ala Lys Tyr Asn Glu Lys Phe Gly 1 5 10 15 <210> 120 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 120 Tyr Ile Asp Pro Tyr Asn Ser Gly Ala Lys Tyr Asn Glu Lys Phe Gly 1 5 10 15 <210> 121 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 121 Tyr Ile Asp Pro Tyr Asn Tyr Gly Ala Lys Tyr Asn Glu Lys Phe Gly 1 5 10 15 <210> 122 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 122 Tyr Ile Asp Pro Tyr Asn Val Gly Ala Lys Tyr Asn Glu Lys Phe Gly 1 5 10 15 <210> 123 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 123 Tyr Ile Asp Pro Tyr Asn Asp Arg Ala Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly      <210> 124 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 124 Tyr Ile Asp Pro Tyr Asn Asp Asn Ala Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly      <210> 125 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 125 Tyr Ile Asp Pro Tyr Asn Asp Gln Ala Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly      <210> 126 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 126 Tyr Ile Asp Pro Tyr Asn Asp Glu Ala Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly      <210> 127 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 127 Tyr Ile Asp Pro Tyr Asn Asp Leu Ala Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly      <210> 128 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 128 Tyr Ile Asp Pro Tyr Asn Asp Lys Ala Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly      <210> 129 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 129 Tyr Ile Asp Pro Tyr Asn Asp Ser Ala Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly      <210> 130 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 130 Tyr Ile Asp Pro Tyr Asn Asp Tyr Ala Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly      <210> 131 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 131 Tyr Ile Asp Pro Tyr Asn Asp Val Ala Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly      <210> 132 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 132 Ala Ala Lys Thr Leu Ala Glu 1 5 <210> 133 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <133> 133 Tyr Ala Lys Thr Leu Ala Glu 1 5 <210> 134 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 134 Trp Ala Lys Thr Leu Ala Glu 1 5 <210> 135 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 135 Ser Ala Lys Thr Leu Ala Glu 1 5 <210> 136 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 136 Thr Ala Lys Thr Leu Ala Glu 1 5 <210> 137 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 137 Ile Ala Lys Thr Leu Ala Glu 1 5 <210> 138 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 138 Val Ala Lys Thr Leu Ala Glu 1 5 <139> <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 139 Asn Asn Lys Thr Leu Ala Glu 1 5 <210> 140 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 140 Asn Ile Lys Thr Leu Ala Glu 1 5 <210> 141 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 141 Asn Leu Leu Thr Leu Ala Glu 1 5 <210> 142 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 142 Asn Thr Lys Thr Leu Ala Glu 1 5 <210> 143 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 143 Asn Val Lys Thr Leu Ala Glu 1 5 <210> 144 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 144 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr             20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile         35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Ser Gln Lys Phe     50 55 60 Gln Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Gly Pro Tyr Gly Trp Tyr Phe Asp Val Trp Gly Gln Gly             100 105 110 Thr Thr Val Thr Val Ser Ser         115 <210> 145 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 145 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr             20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile         35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Ser Gln Lys Phe     50 55 60 Gln Gly Arg Val Thr Leu Thr Ser Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Gly Pro Tyr Gly Trp Tyr Phe Asp Val Trp Gly Gln Gly             100 105 110 Thr Thr Val Thr Val Ser Ser         115 <210> 146 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 146 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr             20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Ala Gln Lys Phe     50 55 60 Gln Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Gly Pro Tyr Gly Trp Tyr Phe Asp Val Trp Gly Gln Gly             100 105 110 Thr Thr Val Thr Val Ser Ser         115 <210> 147 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 147 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr             20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Ala Gln Lys Phe     50 55 60 Gln Gly Arg Val Thr Leu Thr Ser Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Gly Pro Tyr Gly Trp Tyr Phe Asp Val Trp Gly Gln Gly             100 105 110 Thr Thr Val Thr Val Ser Ser         115 <210> 148 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 148 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr             20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Ala Gln Lys Phe     50 55 60 Gln Gly Arg Val Thr Leu Thr Ser Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Gly Pro Tyr Gly Trp Tyr Phe Asp Val Trp Gly Gln Gly             100 105 110 Thr Thr Val Thr Val Ser Ser         115 <210> 149 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 149 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr             20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Ala Gln Lys Phe     50 55 60 Gln Gly Arg Val Thr Leu Thr Ser Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Val Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Gly Pro Tyr Gly Trp Tyr Phe Asp Val Trp Gly Gln Gly             100 105 110 Thr Thr Val Thr Val Ser Ser         115 <210> 150 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 150 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu His Ile Tyr Ser Tyr             20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Phe Gly Ser Pro Leu                 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys             100 105 <210> 151 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 151 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu His Ile Tyr Ser Tyr             20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Phe Gly Ser Pro Leu                 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys             100 105 <210> 152 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 152 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu His Ile Tyr Ser Tyr             20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile         35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln His His Phe Gly Ser Pro Leu                 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys             100 105 <210> 153 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 153 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu His Ile Tyr Ser Tyr             20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile         35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln His His Phe Gly Ser Pro Leu                 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys             100 105 <210> 154 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 154 Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly      <210> 155 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 155 Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Ser Gln Lys Phe Gln 1 5 10 15 Gly      <210> 156 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 156 Gln Val Gln Leu Met Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Pro Leu Thr Ser Tyr             20 25 30 Thr Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Ala Ile Trp Ser Ser Gly Ser Thr Asp Tyr Asn Ser Ala Leu Lys     50 55 60 Ser Arg Leu Asn Ile Asn Arg Asp Ser Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Glu Asp Thr Ala Ile Tyr Phe Cys Thr                 85 90 95 Lys Ser Gly Trp Ala Phe Phe Asp Tyr Trp Gly Gln Gly Val Met Val             100 105 110 Thr Val Ser Ser         115 <210> 157 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 157 Asp Ile Gln Met Thr Gln Ser Pro Ser Leu Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Leu Asn Cys Ile Ala Ser Gln Asn Ile Tyr Lys Ser             20 25 30 Leu Ala Trp Tyr Gln Leu Lys Leu Gly Glu Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Asn Ala Asn Ser Leu Gln Ala Gly Ile Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Ala Leu Thr Ile Ser Gly Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Tyr Ser Gly Gly Tyr Thr                 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys             100 105 <210> 158 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 158 Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Lys Ile Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr             20 25 30 Tyr Met Ala Trp Val Arg Leu Ala Pro Gln Lys Gly Leu Glu Trp Val         35 40 45 Ala Ser Ile Ser Tyr Glu Gly Ser Arg Thr His Tyr Gly Asp Ser Val     50 55 60 Arg Gly Arg Phe Ile Ile Ser Arg Asp Asn Pro Lys Asn Ile Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Gly Ser Glu Asp Thr Ala Thr Tyr Phe Cys                 85 90 95 Ala Arg His Thr Gly Thr Leu Asp Trp Leu Val Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Ile Val Ser Ser         115 <210> 159 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 159 Asn Ile Val Met Ala Gln Ser Pro Lys Ser Met Ser Ile Ser Ala Gly 1 5 10 15 Asp Arg Val Thr Met Asn Cys Lys Ala Ser Gln Asn Val Asp Asn Asn             20 25 30 Ile Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile         35 40 45 Phe Tyr Ala Ser Asn Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly     50 55 60 Gly Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Lys Ser Val Gln Ala 65 70 75 80 Glu Asp Ala Ala Phe Tyr Tyr Cys Gln Arg Ile Tyr Asn Phe Pro Thr                 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 160 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> protein <220> <221> MISC_FEATURE (222) (13) .. (13) <223> protein <220> <221> MISC_FEATURE (222) (13) .. (13) <223> protein <400> 160 Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Xaa Glu Gly Tyr 1 5 10 15 Phe Asp Phe              <210> 161 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <220> <221> MISC_FEATURE (222) (3) .. (3) <223> protein <220> <221> MISC_FEATURE (222) (4) .. (4) <223> protein <400> 161 Tyr Ala Xaa Xaa Leu Gln Thr 1 5 <210> 162 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> protein <220> <221> MISC_FEATURE (222) (7) .. (7) <223> protein <400> 162 Gln Gln Tyr Tyr Ser Gly Xaa Thr 1 5 <210> 163 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <220> <221> MISC_FEATURE (222) (6) .. (6) <223> protein <220> <221> MISC_FEATURE (222) (9) .. (9) <223> protein <220> <221> MISC_FEATURE (222) (12) .. (12) <223> protein <220> <221> MISC_FEATURE (222) (15) .. (15) <223> protein <220> <221> MISC_FEATURE <222> (16) .. (16) <223> protein <220> <221> MISC_FEATURE (222) (17) .. (17) <223> protein <220> <221> MISC_FEATURE (222) (23) .. (23) <223> protein <220> <221> MISC_FEATURE (222) (25) .. (25) <223> protein <220> <221> MISC_FEATURE <222> (27) .. (27) <223> protein <220> <221> MISC_FEATURE (222) (30) .. (30) <223> protein <220> <221> MISC_FEATURE (222) (37) .. (37) <223> protein <220> <221> MISC_FEATURE (222) (39) .. (39) <223> protein <220> <221> MISC_FEATURE (222) (40) .. (40) <223> protein <220> <221> MISC_FEATURE (222) (43) .. (43) <223> protein <220> <221> MISC_FEATURE (222) (44) .. (44) <223> protein <220> <221> MISC_FEATURE (222) (45) .. (45) <223> protein <220> <221> MISC_FEATURE (222) (48) .. (48) <223> protein <220> <221> MISC_FEATURE (222) (67) .. (67) <223> protein <220> <221> MISC_FEATURE (222) (68) .. (68) <223> protein <220> <221> MISC_FEATURE (222) (70) .. (70) <223> protein <220> <221> MISC_FEATURE (222) (71) .. (71) <223> protein <220> <221> MISC_FEATURE <222> (79) .. (79) <223> protein <220> <221> MISC_FEATURE <222> (81) .. (81) <223> protein <220> <221> MISC_FEATURE (222) (83) .. (83) <223> protein <220> <221> MISC_FEATURE (222) (87) .. (87) <223> protein <220> <221> MISC_FEATURE <222> (88) .. (88) <223> protein <220> <221> MISC_FEATURE (222) (92) .. (92) <223> protein <220> <221> MISC_FEATURE (222) (94) .. (94) <223> protein <220> <221> MISC_FEATURE <222> (110) .. (110) <223> protein <220> <221> MISC_FEATURE (222) (119) .. (119) <223> protein <220> <221> MISC_FEATURE (222) (122) .. (122) <223> protein <220> <221> MISC_FEATURE <222> (123) .. (123) <223> protein <400> 163 Glu Val Gln Leu Gln Xaa Ser Gly Xaa Gly Leu Xaa Lys Pro Xaa Xaa 1 5 10 15 Xaa Leu Ser Leu Thr Cys Xaa Val Xaa Gly Xaa Ser Ile Xaa Ser Asp             20 25 30 Tyr Trp Gly Trp Xaa Arg Xaa Xia Pro Gly Xaa Xaa Xaa Glu Trp Xaa         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Xaa Xaa Ile Xaa Xaa Asp Thr Ser Lys Asn Gln Phe Xaa Leu 65 70 75 80 Xaa Leu Xaa Ser Val Thr Xaa Xaa Asp Thr Ala Xaa Tyr Xaa Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Xaa Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Xaa Gly Thr Xaa Xaa Thr Val Ser Ser         115 120 125 <210> 164 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> protein <220> <221> MISC_FEATURE (222) (6) .. (6) <223> protein <220> <221> MISC_FEATURE (222) (9) .. (9) <223> protein <220> <221> MISC_FEATURE (222) (12) .. (12) <223> protein <220> <221> MISC_FEATURE (222) (15) .. (15) <223> protein <220> <221> MISC_FEATURE <222> (16) .. (16) <223> protein <220> <221> MISC_FEATURE (222) (23) .. (23) <223> protein <220> <221> MISC_FEATURE (222) (25) .. (25) <223> protein <220> <221> MISC_FEATURE <222> (27) .. (27) <223> protein <220> <221> MISC_FEATURE (222) (30) .. (30) <223> protein <220> <221> MISC_FEATURE (222) (37) .. (37) <223> protein <220> <221> MISC_FEATURE (222) (39) .. (39) <223> protein <220> <221> MISC_FEATURE (222) (40) .. (40) <223> protein <220> <221> MISC_FEATURE (222) (44) .. (44) <223> protein <220> <221> MISC_FEATURE (222) (45) .. (45) <223> protein <220> <221> MISC_FEATURE (222) (48) .. (48) <223> protein <220> <221> MISC_FEATURE (222) (67) .. (67) <223> protein <220> <221> MISC_FEATURE (222) (71) .. (71) <223> protein <220> <221> MISC_FEATURE (222) (83) .. (83) <223> protein <220> <221> MISC_FEATURE <222> (88) .. (88) <223> protein <220> <221> MISC_FEATURE <222> (110) .. (110) <223> protein <220> <221> MISC_FEATURE (222) (122) .. (122) <223> protein <220> <221> MISC_FEATURE <222> (123) .. (123) <223> protein <400> 164 Glu Val Gln Leu Gln Xaa Ser Gly Xaa Gly Leu Xaa Lys Pro Xaa Xaa 1 5 10 15 Thr Leu Ser Leu Thr Cys Xaa Val Xaa Gly Xaa Ser Ile Xaa Ser Asp             20 25 30 Tyr Trp Gly Trp Xaa Arg Xaa Pro Gly Lys Xaa Xaa Glu Trp Xaa         35 40 45 Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys     50 55 60 Ser Arg Xaa Thr Ile Ser Xaa Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Xaa Ser Val Thr Ala Xaa Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Xaa Glu Gly             100 105 110 Tyr Phe Asp Phe Trp Gly Gln Gly Thr Xaa Xaa Thr Val Ser Ser         115 120 125 <210> 165 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> protein <220> <221> MISC_FEATURE (222) (10) .. (10) <223> protein <220> <221> MISC_FEATURE (222) (21) .. (21) <223> protein <220> <221> MISC_FEATURE (222) (22) .. (22) <223> protein <220> <221> MISC_FEATURE (222) (40) .. (40) <223> protein <220> <221> MISC_FEATURE (222) (42) .. (42) <223> protein <220> <221> MISC_FEATURE (222) (43) .. (43) <223> protein <220> <221> MISC_FEATURE <222> (46) .. (46) <223> protein <220> <221> MISC_FEATURE (222) (52) .. (52) <223> protein <220> <221> MISC_FEATURE (222) (53) .. (53) <223> protein <220> <221> MISC_FEATURE <222> (58) .. (58) <223> protein <220> <221> MISC_FEATURE (222) (77) .. (77) <223> protein <220> <221> MISC_FEATURE (222) (83) .. (83) <223> protein <220> <221> MISC_FEATURE (222) (87) .. (87) <223> protein <220> <221> MISC_FEATURE (222) (95) .. (95) <223> protein <220> <221> MISC_FEATURE (222) (105) .. (105) <223> protein <400> 165 Asp Ile Gln Met Thr Gln Ser Pro Ser Xaa Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Xaa Xaa Cys Lys Ala Ser Gln Ser Ile His Lys Asn             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Xaa Gly Xaa Xaa Pro Lys Xaa Leu Ile         35 40 45 Tyr Tyr Ala Xaa Xaa Leu Gln Thr Gly Xaa Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Xaa Leu Gln Pro 65 70 75 80 Glu Asp Xaa Ala Thr Tyr Xaa Cys Gln Gln Tyr Tyr Ser Gly Xaa Thr                 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Xaa Lys             100 105 <210> 166 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> protein <220> <221> misc_feature (222) (43) .. (43) <223> protein <220> <221> misc_feature <222> (46) .. (46) <223> protein <220> <221> misc_feature (222) (52) .. (52) <223> protein <220> <221> MISC_FEATURE (222) (53) .. (53) <223> protein <220> <221> MISC_FEATURE <222> (56) .. (56) <223> protein <220> <221> misc_feature <222> (58) .. (58) <223> protein <220> <221> MISC_FEATURE (222) (62) .. (62) <223> protein <220> <221> MISC_FEATURE (222) (63) .. (63) <223> protein <220> <221> MISC_FEATURE (222) (68) .. (68) <223> protein <220> <221> MISC_FEATURE (222) (83) .. (83) <223> protein <220> <221> MISC_FEATURE (222) (95) .. (95) <223> protein <400> 166 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Xaa Pro Lys Xaa Leu Ile         35 40 45 Tyr Tyr Ala Xaa Xaa Leu Gln Thr Gly Xaa Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Xaa Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Xaa Thr                 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 167 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> protein <220> <221> MISC_FEATURE (222) (7) .. (7) <223> protein <220> <221> MISC_FEATURE (222) (8) .. (8) <223> protein <220> <221> MISC_FEATURE (222) (12) .. (12) <223> protein <220> <221> MISC_FEATURE (222) (13) .. (13) <223> protein <220> <221> MISC_FEATURE <222> (16) .. (16) <223> protein <400> 167 Tyr Ile Asp Pro Tyr Asn Xaa Xaa Ala Lys Tyr Xaa Xaa Lys Phe Xaa 1 5 10 15 Gly      <210> 168 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <220> <221> misc_feature (222) (1) .. (1) <223> protein <220> <221> misc_feature (222) (2) .. (2) <223> protein <400> 168 Xaa Xaa Lys Thr Leu Ala Glu 1 5 <210> 169 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <220> <221> MISC_FEATURE (222) (5) .. (5) <223> protein <220> <221> MISC_FEATURE (222) (9) .. (9) <223> protein <220> <221> MISC_FEATURE <222> (11) .. (11) <223> protein <220> <221> MISC_FEATURE (222) (12) .. (12) <223> protein <220> <221> MISC_FEATURE <222> (16) .. (16) <223> protein <220> <221> MISC_FEATURE (222) (20) .. (20) <223> protein <220> <221> MISC_FEATURE (222) (38) .. (38) <223> protein <220> <221> MISC_FEATURE (222) (40) .. (40) <223> protein <220> <221> MISC_FEATURE (222) (44) .. (44) <223> protein <220> <221> MISC_FEATURE <222> (56) .. (56) <223> protein <220> <221> MISC_FEATURE (222) (57) .. (57) <223> protein <220> <221> MISC_FEATURE (222) (61) .. (61) <223> protein <220> <221> MISC_FEATURE (222) (62) .. (62) <223> protein <220> <221> MISC_FEATURE (222) (65) .. (65) <223> protein <220> <221> MISC_FEATURE (222) (67) .. (67) <223> protein <220> <221> MISC_FEATURE (222) (68) .. (68) <223> protein <220> <221> MISC_FEATURE (222) (72) .. (72) <223> protein <220> <221> MISC_FEATURE <222> (74) .. (74) <223> protein <220> <221> MISC_FEATURE <222> (76) .. (76) <223> protein <220> <221> MISC_FEATURE <222> (79) .. (79) <223> protein <220> <221> MISC_FEATURE (222) (85) .. (85) <223> protein <220> <221> MISC_FEATURE (222) (87) .. (87) <223> protein <220> <221> MISC_FEATURE (222) (89) .. (89) <223> protein <220> <221> MISC_FEATURE <222> (91) .. (91) <223> protein <220> <221> MISC_FEATURE (222) (92) .. (92) <223> protein <220> <221> MISC_FEATURE <222> (104) .. (104) <223> protein <220> <221> MISC_FEATURE <222> (111) .. (111) <223> protein <400> 169 Glu Val Gln Leu Xaa Gln Ser Gly Xaa Glu Xaa Xaa Lys Pro Gly Xaa 1 5 10 15 Ser Val Lys Xaa Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr             20 25 30 Val Met His Trp Val Xaa Gln Xaa Pro Gly Gln Xaa Leu Glu Trp Ile         35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Xaa Xaa Ala Lys Tyr Xaa Xaa Lys Phe     50 55 60 Xaa Gly Xaa Xaa Thr Leu Thr Xaa Asp Xaa Ser Xaa Ser Thr Xaa Tyr 65 70 75 80 Met Glu Leu Ser Xaa Leu Xaa Ser Xaa Asp Xaa Xaa Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Gly Pro Tyr Gly Xaa Tyr Phe Asp Val Trp Gly Xaa Gly             100 105 110 Thr Thr Val Thr Val Ser Ser         115 <210> 170 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <220> <221> MISC_FEATURE <222> (16) .. (16) <223> protein <220> <221> MISC_FEATURE (222) (44) .. (44) <223> protein <220> <221> MISC_FEATURE <222> (56) .. (56) <223> protein <220> <221> MISC_FEATURE (222) (57) .. (57) <223> protein <220> <221> MISC_FEATURE (222) (61) .. (61) <223> protein <220> <221> MISC_FEATURE (222) (62) .. (62) <223> protein <220> <221> MISC_FEATURE (222) (65) .. (65) <223> protein <220> <221> MISC_FEATURE (222) (72) .. (72) <223> protein <220> <221> MISC_FEATURE <222> (74) .. (74) <223> protein <220> <221> MISC_FEATURE <222> (76) .. (76) <223> protein <220> <221> MISC_FEATURE <222> (79) .. (79) <223> protein <220> <221> MISC_FEATURE (222) (85) .. (85) <223> protein <220> <221> MISC_FEATURE (222) (89) .. (89) <223> protein <220> <221> MISC_FEATURE (222) (92) .. (92) <223> protein <220> <221> MISC_FEATURE <222> (104) .. (104) <223> protein <400> 170 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Xaa 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr             20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Xaa Leu Glu Trp Ile         35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Xaa Xaa Ala Lys Tyr Xaa Xaa Lys Phe     50 55 60 Xaa Gly Arg Val Thr Leu Thr Xaa Asp Xaa Ser Xaa Ser Thr Xaa Tyr 65 70 75 80 Met Glu Leu Ser Xaa Leu Arg Ser Xaa Asp Thr Xaa Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Gly Pro Tyr Gly Xaa Tyr Phe Asp Val Trp Gly Gln Gly             100 105 110 Thr Thr Val Thr Val Ser Ser         115 <210> 171 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> protein <220> <221> MISC_FEATURE (222) (9) .. (9) <223> protein <220> <221> MISC_FEATURE (222) (17) .. (17) <223> protein <220> <221> MISC_FEATURE (222) (18) .. (18) <223> protein <220> <221> MISC_FEATURE (222) (40) .. (40) <223> protein <220> <221> MISC_FEATURE (222) (43) .. (43) <223> protein <220> <221> MISC_FEATURE (222) (45) .. (45) <223> protein <220> <221> MISC_FEATURE (222) (48) .. (48) <223> protein <220> <221> MISC_FEATURE (222) (50) .. (50) <223> protein <220> <221> MISC_FEATURE (222) (51) .. (41) <223> protein <220> <221> misc_feature (222) (51) .. (51) <223> protein <220> <221> MISC_FEATURE (222) (70) .. (70) <223> protein <220> <221> MISC_FEATURE (222) (72) .. (72) <223> protein <220> <221> MISC_FEATURE <222> (74) .. (74) <223> protein <220> <221> MISC_FEATURE <222> (76) .. (76) <223> protein <220> <221> MISC_FEATURE (222) (83) .. (83) <223> protein <220> <221> MISC_FEATURE (222) (84) .. (84) <223> protein <220> <221> MISC_FEATURE <222> (100) .. (100) <223> protein <220> <221> MISC_FEATURE <222> (103) .. (103) <223> protein <220> <221> MISC_FEATURE <222> (106) .. (106) <223> protein <400> 171 Asp Ile Gln Met Thr Gln Ser Pro Xaa Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Xaa Xaa Val Thr Ile Thr Cys Arg Ala Ser Glu His Ile Tyr Ser Tyr             20 25 30 Leu Ser Trp Tyr Gln Gln Lys Xaa Gly Lys Xaa Pro Xaa Leu Leu Xaa         35 40 45 Tyr Xaa Xaa Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Xaa Phe Xaa Leu Xaa Ile Xaa Ser Leu Gln Pro 65 70 75 80 Glu Asp Xaa Xaa Thr Tyr Tyr Cys Gln His His Phe Gly Ser Pro Leu                 85 90 95 Thr Phe Gly Xaa Gly Thr Xaa Leu Glu Xaa Lys             100 105 <210> 172 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> protein <220> <221> MISC_FEATURE (222) (43) .. (43) <223> protein <220> <221> MISC_FEATURE (222) (50) .. (50) <223> protein <220> <221> MISC_FEATURE (222) (51) .. (51) <223> protein <220> <221> MISC_FEATURE (222) (70) .. (70) <223> protein <220> <221> MISC_FEATURE (222) (83) .. (83) <223> protein <400> 172 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu His Ile Tyr Ser Tyr             20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Xaa Pro Lys Leu Leu Ile         35 40 45 Tyr Xaa Xaa Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Xaa Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Xaa Ala Thr Tyr Tyr Cys Gln His His Phe Gly Ser Pro Leu                 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys             100 105 <210> 173 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> protein <220> <221> MISC_FEATURE (222) (6) .. (6) <223> protein <400> 173 Gly Gly Pro Tyr Gly Xaa Tyr Phe Asp Val 1 5 10 <210> 174 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 174 Gly Gly Pro Tyr Gly Ala Tyr Phe Asp Val 1 5 10 <175> 175 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 175 Gly Gly Pro Tyr Gly Asp Tyr Phe Asp Val 1 5 10 <210> 176 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 176 Gly Gly Pro Tyr Gly Glu Tyr Phe Asp Val 1 5 10 <210> 177 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 177 Gly Gly Pro Tyr Gly Phe Tyr Phe Asp Val 1 5 10 <210> 178 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 178 Gly Gly Pro Tyr Gly Gly Tyr Phe Asp Val 1 5 10 <210> 179 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 179 Gly Gly Pro Tyr Gly Ile Tyr Phe Asp Val 1 5 10 <210> 180 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 180 Gly Gly Pro Tyr Gly Lys Tyr Phe Asp Val 1 5 10 <210> 181 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 181 Gly Gly Pro Tyr Gly Asn Tyr Phe Asp Val 1 5 10 <210> 182 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 182 Gly Gly Pro Tyr Gly Gln Tyr Phe Asp Val 1 5 10 <210> 183 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 183 Gly Gly Pro Tyr Gly Arg Tyr Phe Asp Val 1 5 10 <210> 184 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 184 Gly Gly Pro Tyr Gly Ser Tyr Phe Asp Val 1 5 10 <210> 185 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 185 Gly Gly Pro Tyr Gly Thr Tyr Phe Asp Val 1 5 10 <210> 186 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 186 Gly Gly Pro Tyr Gly Val Tyr Phe Asp Val Ser Glu Gln Ile Asp 1 5 10 15 <210> 187 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 187 Gly Gly Pro Tyr Gly Tyr Tyr Phe Asp Val 1 5 10 <210> 188 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 188 Gly Ala Trp Met Asp 1 5 <210> 189 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 189 Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu Ser 1 5 10 15 Val Lys Gly              <210> 190 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 190 Ala Leu Tyr Asp Gly Phe Tyr Phe Asp Tyr 1 5 10 <210> 191 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 191 Ser Ala Ser Ser Ser Val Ser Ser Gly Tyr Leu Tyr 1 5 10 <210> 192 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 192 Gly Thr Ser Thr Leu Ala Ser 1 5 <210> 193 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 193 His Gln Trp Ser Ser Phe Pro Tyr Thr 1 5 <210> 194 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 194 Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Gly Ala             20 25 30 Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val         35 40 45 Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu     50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Ser Gly Ile Tyr                 85 90 95 Tyr Cys Arg Gly Ala Leu Tyr Asp Gly Phe Tyr Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Thr Leu Thr Val Ser Ser         115 120 <210> 195 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 195 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Asn Leu Thr Cys Ser Ala Ser Ser Ser Val Ser Ser Gly             20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp         35 40 45 Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Val Pro Ala Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Asn Met Glu 65 70 75 80 Ala Glu Asp Ala Ala Ser Tyr Phe Cys His Gln Trp Ser Ser Phe Pro                 85 90 95 Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys             100 105 <210> 196 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> protein <220> <221> MISC_FEATURE (222) (33) .. (33) <223> protein <220> <221> MISC_FEATURE (222) (34) .. (34) <223> protein <220> <221> MISC_FEATURE <222> (76) .. (76) <223> protein <220> <221> MISC_FEATURE <222> (79) .. (79) <223> protein <220> <221> MISC_FEATURE (222) (80) .. (80) <223> protein <220> <221> MISC_FEATURE (222) (85) .. (85) <223> protein <220> <221> MISC_FEATURE (222) (86) .. (86) <223> protein <220> <221> MISC_FEATURE (222) (87) .. (87) <223> protein <220> <221> MISC_FEATURE (222) (93) .. (93) <223> protein <220> <221> MISC_FEATURE <222> (104) .. (104) <223> protein <220> <221> MISC_FEATURE (222) (105) .. (105) <223> protein <400> 196 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Ala             20 25 30 Xaa Xaa Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu     50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Xaa Ser Lys Xaa Xaa 65 70 75 80 Val Tyr Leu Gln Xaa Xaa Xaa Leu Arg Ala Glu Asp Xaa Ala Val Tyr                 85 90 95 Tyr Cys Arg Gly Ala Leu Tyr Xaa Xaa Phe Tyr Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 197 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 197 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Ala             20 25 30 Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu     50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr                 85 90 95 Tyr Cys Arg Gly Ala Leu Tyr Asp Gly Phe Tyr Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 198 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 198 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Ala             20 25 30 Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu     50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr                 85 90 95 Tyr Cys Arg Gly Ala Leu Tyr Asp Gly Phe Tyr Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 199 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 199 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Ala             20 25 30 Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu     50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr                 85 90 95 Tyr Cys Arg Gly Ala Leu Tyr Asp Gly Phe Tyr Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 200 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> protein <220> <221> MISC_FEATURE (222) (20) .. (20) <223> protein <220> <221> MISC_FEATURE <222> (46) .. (46) <223> protein <220> <221> MISC_FEATURE (222) (48) .. (48) <223> protein <220> <221> MISC_FEATURE (222) (59) .. (59) <223> protein <220> <221> MISC_FEATURE <222> (79) .. (79) <223> protein <220> <221> MISC_FEATURE (222) (84) .. (84) <223> protein <220> <221> MISC_FEATURE (222) (92) .. (92) <223> protein <220> <221> MISC_FEATURE <107> (107) .. (107) <223> protein <400> 200 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Xaa Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly             20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Xaa Leu Xaa         35 40 45 Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Xaa Pro Ala Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Xaa Glu 65 70 75 80 Pro Glu Asp Xaa Ala Val Tyr Tyr Cys His Gln Xaa Ser Ser Phe Pro                 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Xaa Lys             100 105 <210> 201 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 201 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly             20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Trp         35 40 45 Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Val Pro Ala Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Trp Ser Ser Phe Pro                 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 202 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 202 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly             20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp         35 40 45 Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Val Pro Ala Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Trp Ser Ser Phe Pro                 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 203 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 203 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly             20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp         35 40 45 Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Trp Ser Ser Phe Pro                 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 204 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 204 Glu Phe Thr Met His 1 5 <210> 205 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 205 Gly Leu Lys Pro Asp Asn Gly Gly Ile Ser Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly      <206> 206 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 206 Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr 1 5 10 <210> 207 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 207 Lys Ala Ser Gln Asp Val Lys Thr Ala Val Ala 1 5 10 <210> 208 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 208 Ser Ala Ser Tyr Arg Asn Thr 1 5 <210> 209 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 209 Gln Gln His Tyr Ser Thr Pro Phe Thr 1 5 <210> 210 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 210 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Asn Gly Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Ala Val Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Thr Leu Thr Val Ser Ser         115 <210> 211 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 211 Asp Ile Val Leu Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Lys Thr Ala             20 25 30 Val Ala Trp Tyr Gln Gln Lys Ser Gly Gln Ser Pro Lys Leu Leu Ile         35 40 45 Tyr Ser Ala Ser Tyr Arg Asn Thr Gly Val Pro Asp Arg Phe Thr Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Asp Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln His Tyr Ser Thr Pro Phe                 85 90 95 Thr Phe Gly Thr Gly Thr Lys Leu Glu Leu Lys             100 105 <210> 212 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <220> <221> MISC_FEATURE <222> (11) .. (11) <223> protein <220> <221> MISC_FEATURE (222) (12) .. (12) <223> protein <220> <221> misc_feature (222) (24) .. (24) <223> protein <220> <221> MISC_FEATURE (222) (30) .. (30) <223> protein <220> <221> MISC_FEATURE (222) (33) .. (33) <223> protein <220> <221> misc_feature (222) (34) .. (34) <223> protein <220> <221> MISC_FEATURE (222) (37) .. (37) <223> protein <220> <221> misc_feature (222) (38) .. (38) <223> protein <220> <221> MISC_FEATURE (222) (47) .. (47) <223> protein <220> <221> misc_feature (222) (55) .. (56) <223> protein <220> <221> MISC_FEATURE (222) (61) .. (61) <223> protein <220> <221> MISC_FEATURE (222) (62) .. (62) <223> protein <220> <221> misc_feature <222> (74) .. (74) <223> protein <220> <221> misc_feature (222) (77) .. (77) <223> protein <220> <221> misc_feature <222> (81) .. (81) <223> protein <220> <221> misc_feature <222> (91) .. (91) <223> protein <220> <221> misc_feature (222) (105) .. (106) <223> protein <400> 212 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Xaa Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Xaa His Trp Val Xaa Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Xaa Xaa Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Xaa Ser Thr Xaa Thr Ala Tyr 65 70 75 80 Xaa Glu Leu Ser Ser Leu Arg Ser Glu Asp Xaa Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Xaa Xaa Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 213 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 213 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Asn Gly Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 214 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 214 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Asn Gly Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 215 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 215 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Asn Gly Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 216 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> protein <220> <221> MISC_FEATURE <222> (55) .. (55) <223> protein <220> <221> MISC_FEATURE <222> (56) .. (56) <223> protein <220> <221> MISC_FEATURE (222) (60) .. (60) <223> protein <220> <221> MISC_FEATURE (222) (73) .. (73) <223> protein <400> 216 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Lys Thr Ala             20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Ser Ala Ser Tyr Arg Xaa Xaa Gly Val Pro Xaa Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Xaa Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Phe                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 217 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 217 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Lys Thr Ala             20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Ser Ala Ser Tyr Arg Asn Thr Gly Val Pro Asp Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Phe                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 218 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 218 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Lys Thr Ala             20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Ser Ala Ser Tyr Arg Asn Thr Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Phe                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 219 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 219 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Lys Thr Ala             20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Ser Ala Ser Tyr Arg Asn Thr Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Phe                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 220 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 220 Ala Leu Tyr Glu Gly Phe Tyr Phe Asp Tyr 1 5 10 <210> 221 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 221 Ala Leu Tyr Asp Ala Phe Tyr Phe Asp Tyr 1 5 10 <210> 222 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 222 Ala Leu Tyr Asp Ser Phe Tyr Phe Asp Tyr 1 5 10 <210> 223 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 223 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Ala             20 25 30 Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu     50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr                 85 90 95 Tyr Cys Arg Gly Ala Leu Tyr Glu Gly Phe Tyr Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 224 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 224 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Ala             20 25 30 Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu     50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr                 85 90 95 Tyr Cys Arg Gly Ala Leu Tyr Asp Ala Phe Tyr Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 225 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 225 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Ala             20 25 30 Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu     50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr                 85 90 95 Tyr Cys Arg Gly Ala Leu Tyr Asp Ser Phe Tyr Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 226 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 226 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Ala             20 25 30 Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu     50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr                 85 90 95 Tyr Cys Arg Gly Ala Leu Tyr Asp Glu Phe Tyr Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 227 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 227 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Ala             20 25 30 Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu     50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr                 85 90 95 Tyr Cys Arg Gly Ala Leu Tyr Asp Ala Phe Tyr Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 228 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 228 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Ala             20 25 30 Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu     50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr                 85 90 95 Tyr Cys Arg Gly Ala Leu Tyr Asp Ser Phe Tyr Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 229 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 229 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Ala             20 25 30 Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu     50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr                 85 90 95 Tyr Cys Arg Gly Ala Leu Tyr Glu Gly Phe Tyr Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 230 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 230 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Ala             20 25 30 Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu     50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr                 85 90 95 Tyr Cys Arg Gly Ala Leu Tyr Asp Ala Phe Tyr Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 231 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 231 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Ala             20 25 30 Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu     50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr                 85 90 95 Tyr Cys Arg Gly Ala Leu Tyr Asp Ser Phe Tyr Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 232 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 232 His Gln Ala Ser Ser Phe Pro Tyr Thr 1 5 <210> 233 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 233 His Gln Asp Ser Ser Phe Pro Tyr Thr 1 5 <210> 234 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 234 His Gln Glu Ser Ser Phe Pro Tyr Thr 1 5 <210> 235 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 235 His Gln Phe Ser Ser Phe Pro Tyr Thr 1 5 <210> 236 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 236 His Gln Gly Ser Ser Phe Pro Tyr Thr 1 5 <210> 237 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 237 His Gln His Ser Ser Phe Pro Tyr Thr 1 5 <210> 238 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 238 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly             20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Trp         35 40 45 Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Val Pro Ala Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Ala Ser Ser Phe Pro                 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 239 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 239 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly             20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Trp         35 40 45 Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Val Pro Ala Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Asp Ser Ser Phe Pro                 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 240 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 240 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly             20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Trp         35 40 45 Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Val Pro Ala Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Glu Ser Ser Phe Pro                 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 241 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 241 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly             20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Trp         35 40 45 Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Val Pro Ala Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Phe Ser Ser Phe Pro                 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 242 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 242 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly             20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Trp         35 40 45 Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Val Pro Ala Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Gly Ser Ser Phe Pro                 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 243 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 243 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly             20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Trp         35 40 45 Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Val Pro Ala Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln His Ser Ser Phe Pro                 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 244 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 244 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly             20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp         35 40 45 Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Val Pro Ala Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Ala Ser Ser Phe Pro                 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 245 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 245 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly             20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp         35 40 45 Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Val Pro Ala Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Asp Ser Ser Phe Pro                 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 246 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 246 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly             20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp         35 40 45 Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Val Pro Ala Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Glu Ser Ser Phe Pro                 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 247 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 247 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly             20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp         35 40 45 Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Val Pro Ala Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Phe Ser Ser Phe Pro                 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 248 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 248 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly             20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp         35 40 45 Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Val Pro Ala Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Gly Ser Ser Phe Pro                 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 249 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 249 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly             20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp         35 40 45 Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Val Pro Ala Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln His Ser Ser Phe Pro                 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 250 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 250 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly             20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp         35 40 45 Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Ala Ser Ser Phe Pro                 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 251 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 251 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly             20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp         35 40 45 Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Asp Ser Ser Phe Pro                 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 252 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 252 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly             20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp         35 40 45 Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Glu Ser Ser Phe Pro                 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 253 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 253 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly             20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp         35 40 45 Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Phe Ser Ser Phe Pro                 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 254 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 254 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly             20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp         35 40 45 Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Gly Ser Ser Phe Pro                 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 255 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 255 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly             20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp         35 40 45 Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln His Ser Ser Phe Pro                 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 256 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 256 Gly Leu Lys Pro Asp Gln Gly Gly Ile Ser Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly      <210> 257 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 257 Gly Leu Lys Pro Asp Asp Gly Gly Ile Ser Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly      <210> 258 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 258 Gly Leu Lys Pro Asp Asn Ala Gly Ile Ser Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly      <210> 259 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 259 Gly Leu Lys Pro Asp Thr Gly Gly Ile Ser Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly      <210> 260 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 260 Gly Leu Lys Pro Asp Ser Gly Gly Ile Ser Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly      <210> 261 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 261 Gly Leu Lys Pro Asp Gly Gly Gly Ile Ser Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly      <210> 262 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 262 Gly Leu Lys Pro Asp Asn Ser Gly Ile Ser Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly      <210> 263 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 263 Gly Leu Lys Pro Asp Asn Thr Gly Ile Ser Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly      <210> 264 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 264 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Gln Gly Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 265 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 265 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Asp Gly Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 266 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 266 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Asn Ala Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 267 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 267 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Thr Gly Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 268 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 268 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Ser Gly Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 269 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 269 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Gly Gly Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 270 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 270 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Asn Ser Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 271 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 271 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Asn Thr Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 272 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 272 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Gln Gly Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 273 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 273 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Asp Gly Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 274 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 274 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Asn Ala Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 275 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 275 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Thr Gly Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 276 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 276 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Ser Gly Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 277 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 277 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Gly Gly Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 278 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 278 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Asn Ser Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 279 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 279 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Asn Thr Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 280 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 280 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Gln Gly Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 281 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 281 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Asp Gly Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 282 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 282 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Asn Ala Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 283 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 283 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Thr Gly Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 284 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 284 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Ser Gly Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 285 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 285 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Gly Gly Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 286 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 286 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Asn Ser Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 287 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 287 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Glu Phe             20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Gly Gly Leu Lys Pro Asp Asn Thr Gly Ile Ser Tyr Asn Gln Lys Phe     50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 288 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 288 Ser Ser Thr Thr Ala Gln Val Asn Trp Glu Gln Gln Asp Gln Leu 1 5 10 15 <210> 289 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 289 Ile Tyr His Thr Tyr Pro Asp Gly Thr 1 5 <210> 290 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 290 Gly Arg Ile Phe Leu 1 5 <210> 291 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> protein IgG1 <400> 291 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr             20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser         35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser     50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys                 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys             100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro         115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys     130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu                 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu             180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn         195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly     210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr                 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn             260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe         275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn     290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys                 325 330 <210> 292 <211> 325 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 292 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr 1 5 10 15 Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro             20 25 30 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val         35 40 45 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser     50 55 60 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr 65 70 75 80 Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val                 85 90 95 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe             100 105 110 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr         115 120 125 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Asp Val     130 135 140 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 145 150 155 160 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser                 165 170 175 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu             180 185 190 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser         195 200 205 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro     210 215 220 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 225 230 235 240 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala                 245 250 255 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr             260 265 270 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu         275 280 285 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser     290 295 300 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 305 310 315 320 Leu Ser Leu Gly Lys                 325 <210> 293 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> protein <400> 293 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 1 5 10 15 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro             20 25 30 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly         35 40 45 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr     50 55 60 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 65 70 75 80 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val                 85 90 95 Thr Lys Ser Phe Asn Arg Gly Glu Cys             100 105

Claims (30)

(i) about 10 mg / ml to about 200 mg / ml anti-TIGIT antibody, or antigen binding fragment thereof;
(ii) about 5 mM to about 20 mM buffer;
(iii) about 6% weight / volume (w / v) to about 8% (w / v) non-reducing sugar;
(iv) about 0.01% (w / v) to about 0.10% (w / v) nonionic surfactant; And
(v) a formulation comprising about 1 mM to about 20 mM antioxidant. The three light chain CDRs and sequences of claim 1, wherein the anti-TIGIT antibody or antigen-binding fragment thereof comprises CDRL1 of SEQ ID NO 111, CDRL2 of SEQ ID NO 112, CDRL3 of SEQ ID NO 113 3 heavy chain CDRs comprising CDRH1 of SEQ ID NO: 108, CDRH2 of SEQ ID NO: 154, and CDRH3 of SEQ ID NO: 110. 10. The formulation of claim 1, wherein the anti-TIGIT antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 148, and a light chain variable region comprising SEQ ID NO: 152. The antibody of claim 3, wherein the anti-TIGIT antibody comprises (i) a human heavy chain IgG1 constant domain comprising the amino acid sequence of SEQ ID NO: 291 and a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO: 293; Or (ii) a human heavy chain IgG4 constant domain comprising the amino acid sequence of SEQ ID NO: 292 and a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO: 293. The formulation according to any one of claims 1 to 4, wherein the pH of the formulation is 5.3 to 6.2. 6. The formulation according to claim 1, wherein the buffer is L-histidine buffer, the non-reducing sugar is sucrose, the nonionic surfactant is polysorbate 80, the antioxidant is L-methionine, and the formulation is
(i) about 10 mg / ml to about 200 mg / ml anti-TIGIT antibody, or antigen binding fragment thereof;
(ii) about 5 mM to about 20 mM of L-histidine buffer;
(iii) about 6% (w / v) to about 8% (w / v) sucrose;
(iv) about 0.01% (w / v) to about 0.10% (w / v) polysorbate 80; And
(v) about 1 mM to about 20 mM L-methionine. The formulation of claim 1, wherein the formulation comprises from about 8 mM to about 12 mM of L-histidine buffer. 8. The formulation of claim 6 or 7, comprising about 5 mM to about 10 mM L-methionine. The formulation of claim 6, comprising polysorbate 80 at a weight ratio of about 0.02% w / v. The formulation of claim 1, comprising from about 10 mg / ml to about 100 mg / ml of the anti-TIGIT antibody or antigen binding fragment thereof. The formulation of claim 10 wherein the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is about 10 mg / ml, 12.5 mg / ml, 25 mg / ml, 50 mg / ml, 75 mg / ml or 100 mg / ml . The method of claim 1, wherein about 25 mg / mL anti-TIGIT antibody, 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% polysorbate 80, and A formulation comprising about 10 mM L-methionine. The method of claim 1, wherein about 50 mg / mL anti-TIGIT antibody, 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% polysorbate 80, and A formulation comprising about 10 mM L-methionine. The composition of claim 1, wherein about 75 mg / mL anti-TIGIT antibody, 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% polysorbate 80, and A formulation comprising about 10 mM L-methionine. The method of claim 1, wherein about 100 mg / mL anti-TIGIT antibody, 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% polysorbate 80, and A formulation comprising about 10 mM L-methionine. The formulation of claim 1, wherein the formulation has a pH of about 5.5-6.3. The formulation of claim 16, wherein the pH of the formulation is about 5.8-6.0. 18. The formulation according to any one of claims 1 to 17, further comprising an anti-PD1 antibody or antigen binding fragment thereof. The method of claim 18, wherein the anti-human PD-1 antibody or antigen-binding fragment thereof comprises three light chain CDRs of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 and SEQ ID NO: 6, SEQ ID NO: 7 and sequence An agent comprising three heavy chain CDRs of SEQ ID NO: 8. The method according to claim 18 or 19, wherein the anti-human PD-1 antibody or antigen-binding fragment thereof comprises a variable light chain region comprising the amino acid sequence set forth in SEQ ID NO: 4, and an amino acid sequence set forth in SEQ ID NO: 9. Wherein the formulation comprises a variable heavy chain region. The formulation of claim 16, wherein the formulation comprises an anti-human PD-1 antibody that is pembrolizumab. 22. The formulation of any one of claims 18 to 21, wherein the ratio of anti-PD1 antibody to anti-TIGIT antibody is 1: 1. The method of claim 18, wherein about 20 mg / ml anti-PD1 antibody, about 20 mg / ml anti-TIGIT antibody, 10 mM L-histidine buffer, about 7% w / v number A formulation comprising cross, about 0.02% w / v polysorbate 80, and about 10 mM L-methionine. The formulation according to claim 1, further comprising a chelator. The formulation of claim 24, wherein the chelator is DTPA. 26. The formulation according to any one of claims 1 to 25, wherein the formulation is contained in a glass vial or injection device. 27. The formulation according to any one of claims 1 to 26, which is a liquid formulation, frozen below at least -70 [deg.] C., or reconstituted liquid from a lyophilized formulation. The method of claim 1, wherein after 12 months at 5 ° C .:
(i) the monomer (%) of the anti-TIGIT antibody is> 95% as measured by size exclusion chromatography;
(ii) the heavy and light (%) of the anti-TIGIT antibody is> 90% as measured by reduced CE-SDS;
(iii) the heavy and light (%) of the anti-TIGIT antibody is ≧ 95% as measured by reduced CE-SDS;
(iv) the intact IgG (%) of the anti-TIGIT antibody is> 90% as measured by non-reduced CE-SDS;
(v) A formulation wherein the% intact IgG of the anti-TIGIT antibody is ≧ 95% as measured by non-reduced CE-SDS. A method of treating cancer or chronic infection in a human patient in need thereof, comprising administering an effective amount of the agent of any one of claims 1-28. Use of the agent of any one of claims 1 to 28 for the manufacture of a medicament for the treatment of cancer or for the treatment of chronic infection.

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